3.5.2. Osteoblast Growth on the Biomaterials

Ability of the biomaterials to support cell proliferation and growth was assessed by seeding MC3T3-E1 preosteoblasts directly on the biomaterials at low concentration of 3 <sup>×</sup> <sup>10</sup><sup>4</sup> cells/sample. After 72 h of culture, the MC3T3-E1 cells were fixed as described earlier [49] and F-actin cytoskeletal filaments were stained with AlexaFluor635-conjugated phallotoxin (Invitrogen, Carlsbad, California, USA). Cell nuclei were stained using 0.5 µg/mL DAPI (Sigma-Aldrich Chemicals, Warsaw, Poland). Stained cells were observed using CLSM.

#### 3.5.3. Plasma Effect on Proliferation of Stem Cells on the Biomaterials

Human ADSCs were seeded directly on the biomaterials at extremely low concentration of 1.5 <sup>×</sup> <sup>10</sup><sup>4</sup> cells/sample. After 24 h of culture (when the cells were well attached to the biomaterials), the culture medium was discarded and replaced with Hanks' Balanced Salt solution (HBSS, Sigma-Aldrich Chemicals, Warsaw, Poland). Plasma treatment of the samples was performed using GlidArc reactor operated at the atmospheric pressure with the use of nitrogen as a substrate gas. The electrode tips were positioned at a 3 cm distance from the surface of the biomaterials and 7.33 dm<sup>3</sup> /min flow-rate of nitrogen was applied. ADSCs were exposed to nitrogen plasma for 16 s and left for 3 h in HBSS after treatment. Stem cells cultured on the biomaterials and maintained for 3 h in HBSS without plasma treatment served as control samples. Then, HBSS was discarded, fresh complete culture medium was added, and the cells were cultured for further 6 days with medium renewal on the 3rd day. On the 1st and 6th day, stem cells were fixed and stained as described in Section 3.5.2. The number of cells on the surface of the biomaterials was determined by nuclei counting using ImageJ software version 1.52a (Wayne Rasband, National Institutes of Health, Bethesda, Maryland, USA). The doubling time for the stem cells grown on the samples was calculated with the use of Doubling Time Computing software version 3.1.0.

#### 3.5.4. Plasma Effect on Osteogenic Differentiation of Stem Cells on the Biomaterials

Human ADSCs were seeded directly on the biomaterials at high concentration of 5×10<sup>4</sup> cells/sample. After 24 h of culture, the cells were treated with cold nitrogen plasma as described in Section 3.5.3. Three hours after plasma treatment, HBSS was replaced with osteogenic medium (Osteocyte Differentiation Tool, ATCC-LGC standards, Teddington, UK) and the cells were cultured for a further 20 days with half of a medium renewal every 3-4 days. Markers typical of the osteogenic differentiation (Col I, bALP, and OC) were assessed in the cell lysates that were prepared according to the procedure described earlier [46]. The levels of the osteogenic markers were evaluated using human-specific ELISA kits (bALP ELISA Kit, FineTest, Wuhan, China; Collagen alpha-1(I) chain ELISA Kit, EIAab, Wuhan, China; OC ELISA Kit, EIAab, Wuhan, China). The total protein content was also determined for each lysate using BCA Protein Assay Kit (Thermo Fisher Scientific, Waltham, Massachusetts, USA) to normalize the amount of osteogenic markers (ng) per mg of total cellular proteins.

#### *3.6. Statistical Analysis*

Cell culture tests were performed in at least three independent experiments (*n* = 3). Statistical significance was considered at *p* < 0.05 and determined using One-way ANOVA followed by Tukey0 s test (GraphPad Prism 8.0.0 Software, GraphPad Software Inc., California, CA, USA).

## **4. Conclusions**

Within this study it was observed that extrinsic H2O<sup>2</sup> generated by nitrogen plasma and intrinsic H2O<sup>2</sup> generated by the presence of FexOy-free MSNPs in the scaffold positively affected expression of OC gene, but did not compensate the negative effect of MSNPs presence for the expression of bALP and Col I genes. The addition of FexO<sup>y</sup> catalysts in MSNPs always lead to a significant increase of the quantity of reactive species (mainly short-lived species like OH radicals). The presence of these additional short-lived species had a positive effect on bALP and Col I markers but a negative effect on OC gene expression, where the concentration of reactive species was already at its optimum without the presence of the FexO<sup>y</sup> catalysts.

Presented results clearly demonstrated that short-time (16 s) exposure of ADSCs to nitrogen plasma was non-toxic, accelerated proliferation of cells grown on the biomaterial containing FexOy/MSNPs catalyst, and increased OC production by the cells cultured on the scaffold containing MSNPs without FexO<sup>y</sup> decoration. Plasma activation of the biomaterial containing FexOy/MSNPs catalyst resulted in the formation of sufficient amounts of active long-lived species with enhanced local generation of OH radicals, thanks to the FexO<sup>y</sup> catalysts, that had positive impact on stem cell proliferation and at the same time did not negatively affect their osteogenic differentiation. Therefore, plasma-activated FexOy/MSNPs-loaded biomaterial is characterized by improved biocompatibility and has great clinical potential to be used in regenerative medicine applications to improve bone healing process.

#### **Supplementary Materials:** Supplementary materials can be found at http://www.mdpi.com/1422-0067/21/13/ 4738/s1.

**Author Contributions:** Conceptualization, A.P., J.P., C.C., S.H. and D.D.; methodology, A.P., J.P., C.C., S.H. and D.D.; validation, A.P., J.P., M.A., J.-S.T., C.L., M.K. and P.T.; formal analysis, A.P., J.P., M.A., J.-S.T., C.C., S.H. and D.D.; investigation, A.P., J.P., M.A., J.-S.T., C.C., C.L., M.W., M.K. and P.T. and; resources, A.P., J.P., C.C., S.H., D.D. and G.G.; data curation, A.P., J.P., C.C., S.H., D.D. and M.W.; writing—original draft preparation, A.P., J.P., M.A., J.-S.T and C.L.; writing—review and editing, C.C., S.H., and D.D.; visualization, A.P., J.P., M.A., J.-S.T., C.L. and M.W.; supervision, A.P., J.P., C.C., S.H. and D.D.; project administration, C.C., S.H., D.D. and G.G.; funding acquisition, J.P., C.C., S.H., D.D. and G.G. All authors have read and agreed to the published version of the manuscript.

**Funding:** Financial assistance was provided within M-Era.Net 2 transnational research program by National Science Centre in Poland (NCN, project no. UMO-2016/22/Z/ST8/00694), and partially by Fonds National de la Recherche Luxembourg (FNR, Project No. INTER/MERA/16/11454672) and the Belgian Fonds de la Recherche Scientifique-FNRS (F.R.S.-FNRS, Convention No. R.50.13.17.F). The authors acknowledge also the Spanish Government for financial support through Project PCIN-2017-128. CC and CL belong to SGR2017 1165. The paper was developed using the equipment purchased within agreement no. POPW.01.03.00-06-010/09-00 Operational Program Development of Eastern Poland 2007–2013, Priority Axis I, Modern Economy, Operations 1.3. Innovations Promotion.

**Acknowledgments:** We would like to thank for technical support provided by Jean-Francois Statsyns, François Devred (XRD) and Delphine Magnin (SEM-EDX mapping) in Belgium.

**Conflicts of Interest:** The authors declare no conflict of interest.

## **Abbreviations**


