Calcium Carbonate/Gelatin Methacrylate Microspheres for 3D Cell Culture in Bone Tissue Engineering

Hydrogel microspheres have been widely used as cell carriers and three-dimensional cell culture matrices. However, these microspheres are associated with several unfavorable properties for bone tissue engineering applications, for example, their surface is too smooth to attach cells and they do not contain inorganic materials. This article presents a new method to overcome these disadvantages by depositing CaCO₃ crystals on the hydrogel microsphere surface. Specifically, we used a nonplanar flow-focusing microfluidic device to produce gelatin methacrylate (GelMA)-/Na₂CO₃-based microspheres. We subsequently obtained CaCO₃ crystals by a chemical reaction between Na₂CO₃ and CaCl₂. The efficacy of this method was demonstrated by in vitro experiments with human umbilical vein endothelial cells (HUVEC) and immortalized mouse embryonic fibroblasts (iMEF). Cell culture on GelMA/CaCO₃ microspheres showed that cells can easily attach and adhere to GelMA/CaCO₃ microspheres and maintain high viability. Alkaline phosphatase (ALP) expression was increased as well. These results suggest that this novel microsphere has a high potential for bone tissue engineering applications. Impact statement Microspheres as cell culture substrates have attracted a great deal of attention. The combination of organic and inorganic materials offers the unique merits in bone tissue engineering. In this study, there are two contributions. First, the organic and inorganic material of gelatin methacrylate (GelMA) and CaCO₃ were successfully combined, especially, CaCO₃ was formed as crystals to enhance cell attachment. Second, microspheres were successfully fabricated with one-step process: that is, the microfluidic technique was coupled with the CaCO₃ precipitation in situ. Cell culture shows that the GelMA/CaCO₃ microspheres proposed in this study have a high potential for bone tissue engineering applications.