## 4 Discussion  
To properly function within tissues, biomaterials should positively affect cell activity so that they can carry out their physiological role unperturbed, and possibly promote the formation of new tissue, to allow for full integration into the surrounding tissue. In the case of bone substitutes, biomaterials should possess osteeconductive properties, i.e., supporting the formation of new sound bone along their surface. At the very minimum,  
Figure 4: Primary human osteoblasts were cultured in the presence of (**A**,**D**) hydroxyapatite (HA), (**B**,**E**) Sr-substituted HA (SrHA), or (**C**,**F**) magnesium, strontium-substituted HA (MgSrHA) granules. Their viability was then measured using MTT assay, and their protein content was assessed with Lowry assay. Graphs (**A**–**C**) show the difference in viability levels between day three and day seven, expressed as percent change from day three. Graphs (**D**–**F**) show the difference in protein levels between day three and day seven, expressed as percent change from day three. * = \(p\) < 0.05 and **+ = \(p\) < 0.01 vs control. Figure 5: (**A**–**F**): Alkaline phosphatase (ALP) staining: microphotographs of primary human osteoblasts cultured in the presence of complete DMEM enriched with 0.3 mg/mL (**A**,**D**) hydroxyapatite (HA), (**B**,**E**) Sr-substituted HA (SrHA) and (**C**,**F**) magnesium, strontium-substituted HA (MgSrHA) and stained for ALP (red staining). White arrowheads indicate ALP-positive cells in proximity of material granules. Cells were observed at phase contrast. Magnification: 4 X (**A**–**C**), bar = 50 \(\upmu\)m or 10 X (**D**–**F**), bar = 20 \(\upmu\)m. (**G**) Stained cells were counted and plotted as a bar chart. * = \(p\) < 0.05 vs. HA. biomaterials should be bioinert; i.e., they should not elicit undesired cell responses or exert toxic effects on cells. Biocompatibility assessment is, therefore, an important step when developing novel biomaterials [23,24]. In our previous paper [11], we showed the feasibility of sintered MgSr-substituted hydroxyapatite granular materials, and we characterized their physical and chemical properties. The purpose of the present study was to investigate the response of human primary osteoblasts to these materials. The approach that was followed in this paper was inspired by ISO 10993-5 recommendations, the guidelines that focus on biomaterial cytocompatibility [22], albeit with an important exception. Instead of using cell lines, we decided to resort to primary osteoblasts [16]. These cells were already characterized and used in several studies by our group and were shown to display the expression of differentiation markers and the ability to form mineralization nodules in vitro [12,15,25,26]. We believe that such a cell model would increase the transferability of the results to future in vivo investigations, as these cells may more closely resemble tissue osteoblasts than immortalized cells. We therefore proceeded to assess cell viability in the presence of increasing doses of granules. No decrease in cell viability, nor apoptotic cells, was detected in any of the tested samples at any tested concentration, similarly to what was observed with unmodified HA, indicating that the investigated materials did not have toxic effects, as assessed by both MTT assay and protein quantitation. These results were consistent with the literature on HA ceramics, which typically exhibit good cell compatibility [27, 28, 29], and with our cell morphological analysis, which revealed that no signs of distress were observed in cells when cultured in the presence of the conditioned medium. This can be easily interpreted as a lack of release of biologically relevant amounts of cytotoxic compounds from the materials.