Autologous bone transplantation is the gold standard for reconstruction of large bone defects in the fields of orthopedics and odontology. However, limitations such as the grafting mass needed, its availability, and donor site morbidity has led to the search for osteo-inductive biomaterials that possess similar composition and structure to the main inorganic component of bone tissue. In our study, we utilized a bone marrow-derived mesenchymal stem cell (BMSCs) model in 3D to evaluate the osteogenic potential of a novel nanostructured-mesoporous-amorphous biomaterial candidate.
Initial toxicological tests of the novel biomaterial were performed by Live/Dead cell viability assay at various concentrations of the biomaterial.
BMSCs from four healthy donors were mixed with collagen I to achieve 3D structures. These constructs were cultured for 3-5 weeks with or without osteogenic differentiation medium. The groups with no addition of biomaterials were set as negative controls. As positive control, the same setup was used with the addition of hydroxyapatite/β-tricalcium phosphate (HA/βTCP). Finally, our test group contained the novel biomaterial candidate instead.
Osteogenic specific staining (Alizarin red, Von Kossa, Osteoimage®) were used to detect the evidence of mineralization. Osteogenic specific gene expression was analysed by qRT-PCR. In addition, 748 genes involved in various metabolic pathways were quantified by using a single molecule detection system based on fluorescent barcode-hybridization (nCounter®, NanoString).
In this study we introduced an amorphous silica, SiO2, as an additional constituent to calcium phosphate material, thus obtaining a predominantly amorphous material with the composition of CaO-SiO2-P2O5.This material is nanostructured and mesoporous with an average pore size of 13 nm. Toxicology tests proved that the biomaterial is not toxic for BMSCs in concentrations below 10 mg/ml. Histological staining showed clear ossification of gels containing the novel biomaterial even in absence of differentiation medium. The calcification was also more homogeneous compared with the positive control: HA/βTCP. However, qRT-PCR data suggested higher levels of osteogenic related genes in the HA/βTCP group compared with the test group. Multivariate analysis of the 748 metabolic-related genes with nSolver software showed that the novel biomaterial in absence of differentiation medium induced different metabolic pathways compared to the pathways induced by HA/βTCP. No significant difference in gene expression was observed when the groups had been cultured in presence of differentiation medium. Taken together, our results indicate that the novel biomaterial tested in this study, on its own, can induce osteogenesis in BMSCs.
This study shows evidence that this novel nanostructured-mesoporous-amorphous biomaterial has a high potential as a candidate for bone regeneration applications due to its good osteo-inductive capabilities.