Calcium phosphates (CaPs) have been used in bone regeneration for decades. Among the described CaPs, synthetic hydroxyapatite (HAp) has a chemical composition similar to that of natural bone. Gallium-containing compounds have been studied since the 1970s for the treatment of autoimmune diseases and have shown beneficial properties, such as antibacterial activity and inhibition of osteoclast activity. In this study, we synthesized hydroxyapatite (HAp) powder with Ga doping ratios up to 6.9 ± 0.5 wt% using the wet chemical precipitation method. The obtained products were characterized using XRD, BET, FTIR, and ICP-MS. Ga3+ ion release was determined in the cell culture media for up to 30 days. Antibacterial activity was assessed against five bacterial species: Pseudomonas aeruginosa, Escherichia coli, Staphylococcus aureus, Staphylococcus epidermidis, and Streptococcus pyogenes. The biocompatibility of the GaHAp samples was determined in human fibroblasts (hTERT-BJ1) through direct and indirect tests. The structure of the synthesized products was characteristic of HAp, as revealed with XRD and FTIR, although the addition of Ga caused a decrease in the crystallite size. Ga3+ was released from GaHAp paste in a steady manner, with approximately 40% being released within 21 days. GaHAp with the highest gallium contents, 5.5 ± 0.1 wt% and 6.9 ± 0.5 wt%, inhibited the growth of all five bacterial species, with the greatest activity being against Pseudomonas aeruginosa. Biocompatibility assays showed maintained cell viability (~80%) after seven days of indirect exposure to GaHAp. However, when GaHAp with Ga content above 3.3 ± 0.4 wt% was directly applied on the cells, a decrease in metabolic activity was observed on the seventh day. Overall, these results show that GaHAp with Ga content below 3.3 ± 0.4 wt% has attractive antimicrobial properties, without affecting the cell metabolic activity, creating a material that could be used for bone regeneration and prevention of infection.
Different therapeutic strategies for the treatment of non-healing long bone defects have been intensively investigated. Currently used treatments present several limitations that have led to the use of biomaterials in combination with osteogenic growth factors, such as bone morphogenetic proteins (BMPs). Commonly used absorption or encapsulation methods require supra-physiological amounts of BMP2, typically resulting in a so-called initial burst release effect that provokes several severe adverse side effects. A possible strategy to overcome these problems would be to covalently couple the protein to the scaffold. Moreover, coupling should be performed in a site-specific manner in order to guarantee a reproducible product outcome. Therefore, we created a BMP2 variant, in which an artificial amino acid (propargyl-L-lysine) was introduced into the mature part of the BMP2 protein by codon usage expansion (BMP2-K3Plk). BMP2-K3Plk was coupled to functionalized beads through copper catalyzed azide-alkyne cycloaddition (CuAAC). The biological activity of the coupled BMP2-K3Plk was proven in vitro and the osteogenic activity of the BMP2-K3Plk-functionalized beads was proven in cell based assays. The functionalized beads in contact with C2C12 cells were able to induce alkaline phosphatase (ALP) expression in locally restricted proximity of the bead. Thus, by this technique, functionalized scaffolds can be produced that can trigger cell differentiation towards an osteogenic lineage. Additionally, lower BMP2 doses are sufficient due to the controlled orientation of site-directed coupled BMP2. With this method, BMPs are always exposed to their receptors on the cell surface in the appropriate orientation, which is not the case if the factors are coupled via non-site-directed coupling techniques. The product outcome is highly controllable and, thus, results in materials with homogeneous properties, improving their applicability for the repair of critical size bone defects.
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