In situ osteoblast mineralization mediates post-injection mechanical properties of osteoconductive material

The objective of this study was to understand the temporal relationship between in situ generated calcium content (mineralization) and the mechanical properties of an injectable orthobiologic bone-filler material. Murine derived osteoblast progenitor cells were differentiated using osteogenic factors and encapsulated within an injectable polycaprolactone nanofiber-collagen composite scaffold (PN-COL +osteo) to evaluate the effect of mineralization on the mechanical properties of the PN-COL scaffold. A comprehensive study was conducted using both an experimental and a predictive analytical mechanical analysis for mechanical property assessment as well as an extensive in vitro biological analysis for in situ mineralization. Cell proliferation was evaluated using a PicoGreen dsDNA quantification assay and in situ mineralization was analyzed using both an alkaline phosphatase (ALP) assay and an Alizarin Red stain-based assay. Mineralized matrix formation was further evaluated using energy dispersive x-ray spectroscopy (EDS) and visualized using SEM and histological analyses. Compressive mechanical properties of the PN-COL scaffolds were determined using a confined compression stress-relaxation protocol and the obtained data was fit to the standard linear solid viscoelastic material mathematical model to demonstrate a relationship between increased in situ mineralization and the mechanical properties of the PN-COL scaffold. Cell proliferation was constant over the 21 day period. ALP activity and calcium concentration significantly increased at day 14 and 21 as compared to PN-COL -osteo with undifferentiated osteoblast progenitor cells. Furthermore, at day 21 EDS, SEM and von Kossa histological staining confirmed mineralized matrix formation within the PN-COL scaffolds. After 21 days, compressive modulus, peak stress, and equilibrium stress demonstrate significant increases of 3.4-fold, 3.3-fold, and 4.0-fold respectively due to in situ mineralization. Viscoelastic parameters calculated through the standard linear solid mathematical model fit to the stress-relaxation data also indicate improved mechanical properties after in situ mineralization. This investigation clearly demonstrates that in situ mineralization can increase the mechanical properties of an injectable orthobiologic scaffold and can possibly guide the design of an effective osteoconductive injectable material.