*3.4. Determination of Metabolic Activity*

The metabolic activity of hBMSC cells was determined by the MTS assay (Cell Titer96 AQueous One Solution Proliferation Assay; Promega, Germany) at day two after plating. The cell culture medium was replaced by fresh medium containing 10% of MTS dye solution. After 2 h of incubation at 37 ◦C in a humidified CO<sup>2</sup> incubator, 80 µL of medium was transferred into a 96-well plate and the absorbance of the formed formazan dye was measured photometrically at 490 nm.

#### *3.5. Determination of Tissue Non-Specific Alkaline Phosphatase (TNAP) Enzyme Activity*

At day 11 after seeding, hBMSC were analysed for TNAP enzyme activity. TNAP enzyme activity was determined from cell lysates (TNAP lysis buffer: 1.5 M Tris-HCl, pH 10 containing 1 mM ZnCl2, 1 mM MgCl<sup>2</sup> and 1% Triton X-100; Sigma-Aldrich, Germany) with p-nitrophenylphosphate (Sigma-Aldrich, Germany) as a substrate, as previously described [30]. TNAP activity was calculated from a linear calibration curve (r > 0.99) prepared with p-nitrophenolate. Protein concentration of the lysate was determined with RotiQuant protein assay (Roth GmbH, Karlsruhe, Germany) and was calculated from a linear calibration curve (r > 0.99) obtained with bovine serum albumin (Serva, Heidelberg, Germany). Specific TNAP activity is given in mU/mg protein.

#### *3.6. Statistical Analysis*

Cell experiments were performed with cells from three different donors (*n* = 3) each in duplicate. The results were presented as mean ± standard error of the mean (SEOM). Statistical significance was analyzed with GraphPad Prism 8.4 software (Statcon, Witzenhausen, Germany) by ANOVA analysis with Bonferroni's post-test. The contact angle was measured on four water drops for each sample. One-way ANOVA with Tukey's post-test was carried out with the software IBM SPSS Statistics Version 27.

#### **4. Conclusions**

Coating Ti6Al4V with WPI fibrils obtained at pH 2 enriched with heparin and tinzaparin proved to be a successful strategy to create a viable substrate for hBMSC. This substrate for the cells promotes osteogenic differentiation by improving the quality of the differentiated cells, as evidenced by the increase in the TNAP activity. In particular, this work showed that enriching the coating with heparin and tinzaparin improves the aforementioned effect considerably. Specifically, it seems that tinzaparin has the highest impact on the TNAP activity. In any case, further investigations of the molecular mechanism are needed to further elucidate this behavior. As far as the coating protocol is concerned, one hour of adsorption time was sufficient to successfully coat Ti6Al4V substrates. However, the presence of some uncoated areas was detected. Additionally, XPS can be used as a method to evaluate the presence of heparin and proteins on the surface of the material. Nonetheless, it seems that this is not a suitable method for the detection of tinzaparin, and it advisable to use a different approach in future work. The coating increased the hydrophilicity of the material with a higher extent when enriched with tinzaparin compared to heparin. This behavior, together with the statistically significant difference seen between the heparin and tinzaparin coated samples in the TNAP activity test, provides further evidence of the presence of the LMWH molecule.

**Supplementary Materials:** The following are available online at https://www.mdpi.com/article/10 .3390/ijms23010139/s1.

**Author Contributions:** Conceptualization, T.E.L.D.; methodology, T.E.L.D., U.H., L.M. and A.K.; formal analysis, D.F., U.H. and L.M.; investigation, D.F., U.H., L.M., A.M.S. and A.K.; resources, T.E.L.D., U.H., A.M.S., M.A.S. and R.A.S.; writing—original draft preparation, D.F.; writing—review and editing, D.F., U.H., L.M., A.M.S., A.K., M.A.S., R.A.S. and T.E.L.D.; supervision, T.E.L.D., R.A.S. and M.A.S.; project administration, T.E.L.D.; funding acquisition, T.E.L.D., D.F., L.M., A.M.S., R.A.S. and M.A.S. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was funded by EPSRC "A novel coating technology based upon polyatomic ions from plasma" grant number EP/S004505/1 (L.M.)". The University of Milan, Italy, is thanked for financial support for a research stay (D.F.).

**Institutional Review Board Statement:** The study was conducted in accordance with the Declaration of Helsinki, and approved by the Institutional Ethics Committee of TU Dresden (protocol code EK466112016 and approval date 3 November 2016).

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** Not applicable.

**Acknowledgments:** RAS and MAS acknowledge the support from Ministry of Science and Higher Education of the Russian Federation (grant agreement #075-15-2021-588 from 1 June 2021).

**Conflicts of Interest:** The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.

#### **Abbreviations**


#### **References**

