The suitability of hydrogel biomaterials for bone regeneration can be improved by incorporation of an inorganic phase in particle form, thus maintaining hydrogel injectability. In this study, carbonate microparticles containing different amounts of calcium (Ca) and magnesium (Mg) were added to solutions of the anionic polysaccharide gellan gum (GG) to crosslink GG by release of Ca and Mg from microparticles and thereby induce formation of hydrogel-microparticle composites. It was hypothesized that increasing Mg content of microparticles would promote GG hydrogel formation. The effect of Mg incorporation on cytocompatibility and cell growth was also studied. Microparticles were formed by mixing Ca and Mg and [Formula: see text] ions in varying concentrations. Microparticles were characterized physiochemically and subsequently mixed with GG solution to form hydrogel-microparticle composites. The elemental Ca:Mg ratio in the mineral formed was similar to the Ca:Mg ratio of the ions added. In the absence of Mg, vaterite was formed. At low Mg content, magnesian calcite was formed. Increasing the Mg content further caused formation of amorphous mineral. Microparticles of vaterite and magnesium calcite did not induce GG hydrogel formation, but addition of Mg-richer amorphous microparticles induced gelation within 20 min. Microparticles were dispersed homogeneously in hydrogels. MG-63 osteoblast-like cells were cultured in eluate from hydrogel-microparticle composites and on the composites themselves. All composites were cytocompatible. Cell growth was highest on composites containing particles with an equimolar Ca:Mg ratio. In summary, carbonate microparticles containing a sufficient amount of Mg induced GG hydrogel formation, resulting in injectable, cytocompatible hydrogel-microparticle composites.

Mineralization of hydrogel biomaterials is considered desirable to improve their suitability as materials for bone regeneration. Calcium carbonate (CaCO ) has been successfully applied as a bone regeneration material, but hydrogel-CaCO composites have received less attention. Magnesium (Mg) has been used as a component of calcium phosphate biomaterials to stimulate bone-forming cell adhesion and proliferation and bone regeneration in vivo, but its effect as a component of carbonate-based biomaterials remains uninvestigated. In the present study, gellan gum (GG) hydrogels were mineralized enzymatically with CaCO , Mg-enriched CaCO and magnesium carbonate to generate composite biomaterials for bone regeneration. Hydrogels loaded with the enzyme urease were mineralized by incubation in mineralization media containing urea and different ratios of calcium and magnesium ions. Increasing the magnesium concentration decreased mineral crystallinity. At low magnesium concentrations calcite was formed, while at higher concentrations magnesian calcite was formed. Hydromagnesite (Mg (CO ) (OH) .4H O) formed at high magnesium concentration in the absence of calcium. The amount of mineral formed and compressive strength decreased with increasing magnesium concentration in the mineralization medium. The calcium:magnesium elemental ratio in the mineral formed was higher than in the respective mineralization media. Mineralization of hydrogels with calcite or magnesian calcite promoted adhesion and growth of osteoblast-like cells. Hydrogels mineralized with hydromagnesite displayed higher cytotoxicity. In conclusion, enzymatic mineralization of GG hydrogels with CaCO in the form of calcite successfully reinforced hydrogels and promoted osteoblast-like cell adhesion and growth, but magnesium enrichment had no definitive positive effect. Copyright © 2017 John Wiley & Sons, Ltd.

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Mineralization of hydrogels is desirable prior to applications in bone regeneration. CaCO is a widely used bone regeneration material, and Mg, when used as a component of calcium phosphate biomaterials, has promoted bone-forming cell adhesion and proliferation and bone regeneration. In this study, gellan gum hydrogels were mineralized with carbonates containing different amounts of calcium (Ca) and magnesium (Mg) by alternate soaking in, firstly, a calcium and/or magnesium ion solution and, secondly, a carbonate ion solution. This alternate soaking cycle was repeated five times. Five different calcium and/or magnesium ion solutions, containing different molar ratios of Ca to Mg ranging from Mg free to Ca free were compared. Carbonate mineral formed in all sample groups subjected to the alternate soaking cycle. Ca : Mg elemental ratio in the mineral formed was higher than in the respective mineralizing solution. Mineral formed in the absence of Mg was predominantly CaCO in the form of a mixture of calcite and vaterite. Increasing the Mg content in the mineral formed led to the formation of magnesian calcite and decreased the total amount of the mineral formed and its crystallinity. Hydrogel mineralization and increasing Mg content in mineral formed did not obviously improve proliferation of MC3T3-E1 osteoblast-like cells or differentiation after 7 days.

Injectable composites for tissue regeneration can be developed by dispersion of inorganic microparticles and cells in a hydrogel phase. In this study, multifunctional carbonate microparticles containing different amounts of calcium, magnesium and zinc were mixed with solutions of gellan gum (GG), an anionic polysaccharide, to form injectable hydrogel-microparticle composites, containing Zn, Ca and Mg. Zn and Ca were incorporated into microparticle preparations to a greater extent than Mg. Microparticle groups were heterogeneous and contained microparticles of differing shape and elemental composition. Zn-rich microparticles were 'star shaped' and appeared to consist of small crystallites, while Zn-poor, Ca- and Mg-rich microparticles were irregular in shape and appeared to contain lager crystallites. Zn-free microparticle groups exhibited the best cytocompatibility and, unexpectedly, Zn-free composites showed the highest antibacterial activity towards methicilin-resistant Staphylococcus aureus. Composites containing Zn-free microparticles were cytocompatible and therefore appear most suitable for applications as an injectable biomaterial. This study proves the principle of creating bi- and tri-elemental microparticles to induce the gelation of GG to create injectable hydrogel-microparticle composites.