Bone grafting is crucial for treating bone defects, with a growing demand driving the need for effective synthetic substitutes. Hydroxyapatite (HAp) is a common choice, but its brittleness limits its application. The increasing prevalence of implant-associated infections, primarily caused by *Staphylococcus aureus*, *Staphylococcus epidermidis*, and *Pseudomonas aeruginosa*, necessitates strategies beyond antibiotic loading due to rising antibiotic resistance. Magnesium oxide (MgO) offers a compelling alternative, exhibiting broad-spectrum antibacterial activity and promoting bone regeneration through magnesium ion release. This research focuses on developing an HAp/MgO bone substitute with optimized properties, balancing antibacterial effects and biocompatibility.

The literature extensively documents the use of HAp-based bone substitutes due to their biocompatibility and osteointegration. However, their brittleness and susceptibility to infection remain challenges. Studies have explored the antibacterial properties of metal oxides, including MgO, highlighting its cost-effectiveness and broad antibacterial spectrum. Previous work by the authors demonstrated that MgO inclusion reduced bacterial growth and biofilm formation in HAp-based substitutes. Furthermore, the beneficial role of magnesium ions in osteoblast differentiation and proliferation, as well as its involvement in angiogenesis and inflammation, is well-established. The FDA's recognition of MgO as safe further enhances its appeal for bone graft applications. The key challenge is to achieve optimal antibacterial performance while maintaining suitable tissue interactions and biocompatibility, along with sufficient mechanical strength for handling and implantation.

HAp/MgO spherical granules were fabricated using different sintering temperatures (900 °C, 1100 °C, and 1300 °C). Granule characterization involved scanning electron microscopy (SEM) for morphological analysis, X-ray diffraction (XRD) for phase composition, and Fourier-transform infrared spectroscopy (FTIR) for chemical characterization. Mechanical properties were assessed through compression strength and friability tests. Chemical and biodegradation profiles were evaluated using Tris-HCl and simulated body fluid (SBF), respectively, measuring Mg²⁺ release, pH changes, and weight loss. Antibacterial activity was evaluated against *S. aureus*, *S. epidermidis*, and *P. aeruginosa* through planktonic growth and initial adhesion assays. In vitro cytocompatibility was assessed using MC3T3-E1 osteoblasts, measuring metabolic activity and cell adhesion. The in vivo angiogenic and inflammatory potential was evaluated using the chick chorioallantoic membrane (CAM) model. Statistical analysis employed two-way ANOVA, Tukey's and Šidák's multiple comparison tests, Wilcoxon matched-pairs signed rank test, Mann-Whitney test, and Chi-square test.

SEM revealed that higher sintering temperatures led to HAp particle coalescence and larger crystals, reducing nanoporosity. XRD and FTIR confirmed the presence of HAp and MgO, with Mg-O peaks becoming more prominent at higher temperatures. Compression strength significantly increased with sintering temperature, while friability decreased. Mg²⁺ release was inversely proportional to sintering temperature, with lower release observed in SBF compared to Tris-HCl. HAp/MgO granules showed a pH increase in both solutions. Weight loss was highest for granules sintered at 900 °C. Apatite formation was observed in SBF for all but the HAp/MgO granules sintered at 900 °C. The 900 °C and 1100 °C HAp/MgO granules significantly reduced planktonic bacterial growth for all strains tested, except for *P. aeruginosa* at 1300 °C. Initial bacterial adhesion showed a dependence on the bacterial strain and sintering temperature, with MgO generally reducing *P. aeruginosa* adhesion. In vitro studies showed that granules sintered at 900 °C displayed lower osteoblast metabolic activity and cell adhesion, while 1100 °C and 1300 °C granules showed good cytocompatibility. In the CAM assay, HAp/MgO granules significantly increased angiogenesis and decreased inflammation compared to pure HAp granules.

The study successfully demonstrated the tunable properties of the HAp/MgO bone substitute through sintering temperature optimization. The increased mechanical strength at higher temperatures was balanced against potential MgO toxicity. The observed antibacterial activity aligns with established MgO mechanisms, likely involving ROS generation, though the exact contribution of pH and Mg²⁺ release requires further investigation. The decreased antibacterial effect at 1300 °C is attributed to reduced MgO release due to improved material stability. The positive in vivo findings of enhanced angiogenesis and reduced inflammation highlight the beneficial role of Mg²⁺ release. The optimal sintering temperature of 1100 °C achieved a balance between adequate mechanical strength, antibacterial activity, and cytocompatibility.

This study successfully developed an HAp/MgO bone substitute with optimized properties by controlling the sintering temperature. The 1100 °C sintered granules demonstrated a promising balance between antibacterial activity against relevant pathogens, osteoblast cytocompatibility, and enhanced angiogenic response with reduced inflammation. Future work should investigate long-term in vivo efficacy and explore potential improvements in mechanical properties and MgO distribution to further enhance performance.

The study's in vivo assessment was limited to the CAM model, which may not fully replicate the complex environment of bone regeneration in mammals. The duration of in vivo testing was relatively short, necessitating longer-term studies to evaluate long-term biocompatibility and efficacy. Further investigation of the underlying mechanisms of MgO's antibacterial and angiogenic effects is warranted.