Preclinical Development of an Indicator that Specifically Labels Porous Hydroxyapatite

Methods: We selected a protein that had the dual characteristics of (1) high-affinity binding to hydroxyapatite, plus (2) the capacity for chemical modification without loss of this binding activity. The indicator was synthesised by coupling this protein to a blue-black dye using a heterobifunctional cross-linker. Functionality was assessed in vitroby measuring reversible binding to pure hydroxyapatite powder. Preclinical testing included exposure of the indicator to (1) hypomineralised enamel lesions, (2) enamel with experimental demineralisation (phosphoric acid etch), and (3) enamel with natural caries lesions; all followed by water rinsing and photography.  

Results: Successful synthesis was established by showing that the indicator was highly visible and could bind reversibly to pure hydroxyapatite in vitro. Potential clinical utility of the indicator was demonstrated through its ability to highlight the presence of hypomineralised and demineralised enamel regions. Tortuous boundaries of hypomineralised enamel were revealed starkly, so facilitating complete removal of affected tissue. Acid-demineralised enamel was readily visualised in a severity-dependent fashion. Natural caries lesions were rapidly and intensely labelled over most of the visibly-affected regions. However some areas of apparently-intact surface enamel were unlabelled, suggesting that the indicator may usefully distinguish active from inactive caries.

Conclusions: This novel indicator shows promise for delineating hypomineralised enamel from normal, for early detection of caries and enamel erosion, and for distinguishing active from inactive caries. Further work is required to explore each of these aspects, and to investigate the utility of additional reporter elements (e.g. radiographic indicator).