Human Adipose-Derived Mesenchymal Stem Cells to Evaluate the Biological Performances of Ion-doped Sintered Hydroxyapatite Scaffolds

Introduction. Tissue engineering (TE) aims to repair and regenerate tissues damaged by injuries or diseases. For this purpose, bone graft is born as an emerging viable treatment modality. Biomaterials play a key role in these strategies, where they serve as a substrate for the incorporation and release of ions [1-2]. The Mg2+, Sr2+ and Zn2+ ions are active regulators of the proliferation and differentiation of osteoblasts and osteoclasts, thus modulating bone turnover. Specifically, Mg2+ has attracted the attention of the scientific community in the field of TE [3]. In the present work, an in vitro model of human adipose-derived mesenchymal stem cells (hASCs) [4] was used to evaluate the biocompatibility and osteoinductivity properties of four different sets of ion-doped sintered HA powders. The set includes non-doped Sintered Hydroxylapatite (S-HA), as reference material, S-MgHA doped with magnesium, S-MgSrHA doped with magnesium and strontium, and S-MgZnHA doped with zinc and magnesium. Materials and Methods Live/dead dye and confocal laser scanning microscopy were carried out to measure the percentage of living hASC cells. The cellular metabolic activity and the cytoskeleton organization were investigated by AlamarBlue® metabolic assay and Phalloidin-TRITC immunostaining, respectively. The assay was carried out to evaluate the viability of cells attached on the biomaterials (i) S-HA, (ii) S-MgHA, (iii) S-MgSrHA, (iv) S-MgZnHA at day 3, 6 and 9. In order to evaluate the osteogenicity of the four scaffolds, a Real-Time PCR array was performed in order to analyse the expression of genes involved in osteogenic differentiation using the Human Osteogenesis PCR array (GeneGlobe ID-PAHS-026Z, Qiagen, Milan Italy). Cytokine expression (mRNA) is also evaluated in hASC grown on biomaterials. Osteopontin protein (OPN) expression was analysed through immunofluorescence. Results The cytoskeleton architecture of hASCs grown in contact with the scaffolds appeared to be well organized, whereas its integrity remained uninfluenced by the scaffolds. Metabolic activity measured in hASCs grown on the four biomaterials was increased during the experiments, up to day 9. Released ions did not show any cytotoxic effect on hASCs, as revealed by Live/dead staining. Our experiments suggest that the ion-doping scaffolds seem to maintain a positive modulation of osteogenic genes in hASCs compared with the control. The materials also modulate important cytokines. These scaffolds showed high osteoinductivity also revealed by OPN protein expression in cells grown on biomaterials, respect to the control. Discussion The present work suggests that the exploration of different doping agents for scaffolds can yield new sintered materials with optimised biologic performances. Our experiments suggest that the ion-doped scaffolds provide a good microenvironment for hASCs adhesion, viability, and osteoinduction. In addition, our morphology, cell biology and gene expression analyses suggest that hASCs represent an excellent in vitro cellular model to test citocompatibility and osteoinductivity of scaffolds to be used for bone repair/regrowth and tissue engineering. Conclusions Among the materials examined, the magnesium-doped material appears to be cytocompatible, osteoconductive and osteoinductive. Chemical doping with magnesium confirms this ion as ideal for use in scaffolds for bone tissue regenerative medicine. The study also confirms the validity of the resistant cell model as a key model for evaluating materials to be used in the biomedical field.