The influence of mineralization conditions on the effectiveness of enzymatic mineralization of hydrogels

Polysaccharide hydrogels are widely used in food industry and medicine. Gellan gum (GG) recently gained a lot of attention as a promising material for tissue regeneration proposes due to its excellent biocompatibility and similarity to natural extracellular matrix. However, in unmineralized form it is not suitable for bone tissue engineering because of weak mechanical properties. Enzymatic mineralization (e.g. using alkaline phosphatase – ALP) is one of the methods of calcifying of hydrogels and it resembles natural processes occurring during bone healing. The aim of this research was to investigate mineralization of hydrogels and to improve properties of gellan gum scaffolds by adjusting processing conditions. Since ALP does not form with GG covalent bonds, during incubation in mineralization medium (solution of calcium glycerophosphate - CaGP) it is diffusing from the samples. Therefore, mineralization effectiveness depends on the interplay between incoming CaGP and outgoing ALP molecules. We hypothesize that better CaGP availability, especially in the first hours of incubation, can result in more effective and homogenous precipitation of calcium phosphates (CaP) in GG samples. To this end, samples with different GG and ALP concentration were subjected to two different mineralization regimes (more and less frequent CaGP exchanges). We proved that better CaGP availability (more frequent CaGP exchange) resulted in better mechanical properties (Young’s modulus) and more effective mineral formation (higher dry mass percentage) of the samples compared to the same samples mineralized with lower accessibility of CaGP. This may be related to the fact, that in presence of fresh organic substrates, more CaP are formed in the outer parts of the samples at the beginning of the process, that limit ALP diffusion and allow more uniform mineralization.