Biphasic monolithic osteochondral scaffolds obtained by diffusion-limited enzymatic mineralization of gellan gum hydrogel

Biphasic monolithic materials for the treatment of osteochondral defects were produced from polysaccharide hydrogel, gellan gum (GG). GG was enzymatically mineralized by alkaline phosphatase (ALP) in the presence of calcium glycerophosphate (CaGP). The desired distribution of the calcium phosphate (CaP) mineral phase was achieved by limiting the availability of CaGP to specific parts of the GG sample. Therefore, mineralization of GG was facilitated by the diffusion of CaGP, causing the formation of the CaP gradient. The distribution of CaP was analyzed along the cross section of the GG. The formation of a CaP gradient was mainly affected by the mineralization time and the ALP concentration. The formation of CaP was confirmed by Fourier transform infrared spectroscopy (FTIR), Raman spectroscopy and mapping, as well as energy-dispersive X-ray spectroscopy (EDX) mapping of the interphase. The microstructure of mineralized and non-mineralized parts of the material was characterized by scanning electron microscopy (SEM) and atomic force microscopy (AFM) showing sub-micrometer CaP crystal formation, resulting in increased surface roughness. Compression tests and rheometric analyzes showed a 10-fold increase in stiffness of the GG mineralized part. Concomitantly, micromechanical tests performed by AFM showed an increase of Young's modulus from 17.8 to more than 200 kPa. In vitro evaluation of biphasic scaffolds was performed in contact with osteoblast-like MG-63 cells. The mineralized parts of GG were preferentially colonized by the cells over the non-mineralized parts. The results showed that osteochondral scaffolds of the desired structure and properties can be made from GG using a diffusion-limited enzymatic mineralization method.