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Root resorption is an unwanted effect of orthodontic tooth movement. Analysis of dentine proteins in gingival crevicular fluid (GCF) is a potentially safer method of quantifying root resorption compared with conventional radiographic methods. This study aimed to identify and quantify the dentine-specific matrix protein, dentine sialoprotein (DSP), released into GCF during physiological root resorption and orthodontic tooth movement. GCF was collected using micropipettes from 50 second primary molar sites undergoing physiological root resorption in 9- to 14-year olds [coronal group (Rc) with advanced resorption (n=33) and apical group (Ra) with minimal resorption (n=17)] and 20 subjects aged 8–14 years with erupted mandibular second premolars (control group). In addition, GCF was collected from 20 patients undergoing treatment with fixed appliances at two time points, immediately prior to orthodontic intervention (T0) and 12 weeks following commencement of fixed appliance therapy (T1). GCF samples were analysed for DSP using an immunoassay and levels semi-quantified using image analysis. To determine differences between the means of the various experimental and control groups, data based on the relative optical density volumes, were statistically analysed using a parametric t-test.DSP was raised in sites that were undergoing physiological resorption compared with the non-resorbing controls (P < 0.05). Notably, DSP was detected in some control samples. There was no difference in DSP levels for the Rc or Ra groups. DSP was also raised in GCF samples of teeth at 12 weeks following commencement of fixed appliance therapy (P < 0.001). The results highlight the potential for measuring DSP in GCF as a biomarker to monitor root resorption. Dentine is likely to be the major source for DSP in GCF, although alternative origins of bone and cementum are possible.