A nondestructive method for evaluating in vitro osteoblast differentiation on biomaterials using osteoblast-specific fluorescence.

Transgenic mice with a Col1a1-promoter-driven transgene pOBCol2.3GFP were previously developed to visually identify mature osteoblasts through fluorescent expression. Our goal was to determine if this technology could be used to nondestructively evaluate the in vitro differentiation of osteoprogenitor cells on biomaterials such as biomimetic carbonated hydroxyapatite (cHA). Primary osteoprogenitor cells were harvested from calvaria of neonatal Col2.3GFP transgenic mice and cultured on cHA and a tissue culture polystyrene (TCPS) control. The distribution of intensities and area percentage of green fluorescent protein (GFP)-positive cells were quantified using fluorimetry and image analysis of fluorescent microscopy. At 14 days, an increased area and higher mean intensity of GFP-positive cells was observed on cHA as compared to TCPS, indicating more rapid differentiation on cHA. Notably, there were large continuous regions of GFP-positive osteoblasts on cHA, in contrast to the sparse, nodules of osteoblasts on TCPS, implying that cHA provides an osteogenic cue to cells. Xylenol orange staining was capable of distinguishing osteoblast-initiated mineral from the cHA substrate. With this method the unique pattern of osteoblast differentiation on cHA was clearly observed for the first time. Importantly, the generalized method can be used for rapid, high-throughput, nondestructive screening of biomaterials intended to enhance osteogenic differentiation.