The Use of Non-Invasive Biomarkers as Indicators of Skeletal Maturity in Children

Background: In paediatrics and orthodontics the estimation of growth and skeletal maturational level of children is essential for the prediction of future growth potential, diagnosis of developmental deviations and treatment planning. Skeletal maturity indicators identified on hand-wrist radiographs and, lately, on lateral cephalometric radiographs (Cervical Vertebra Maturation - CVM method) are widely used. However, these imaging techniques present several disadvantages and mainly the radiation exposure.

Objective: A simple clinical method is proposed to estimate individual skeletal maturity without using radiation. Biomarkers in saliva and Gingival Crevicular Fluid (GCF), possibly directly involved in bone growth, were investigated.

Methods: Salivary and GCF samples were obtained from children with the use of a pipette and a periopaper strip respectively. Saliva and GCF samples were analysed by an untargeted metabolomics GC-MS method. 0.1 mL of saliva samples were mixed with 0.3 mL of methanol and the supernatant was evaporated, 30 μL MeOX (20 mg/mL) in pyridine were added and incubated for 2h at 60oC, then 10 μL IS in pyridine and 60 μL MSTFA with 1% TMCS were added left for 1h at 60oC. For GCF analysis, one periopaper containing the sample was mixed with 0.5 mL of methanol. The supernatant was evaporated and the same derivatization procedure was followed. Finally, 1 uL was injected in the GC-MS.

Results: Initial results indicate 68 and 44 endogenous metabolites in saliva and GCF, respectively. Several amino acids, observed to differentiate between age groups, are candidate biomarkers for identifying individual skeletal maturity.

Conclusion: Novel molecules and metabolites are investigated as indicators of skeletal maturity in children by performing both saliva and GCF profiling; the common endogenous metabolites are compared. Results are important for paediatrics and orthodontics, not only for growth estimation, but also for accurate direct diagnosis and treatment planning, without radiographs.

This research is co-financed by Greece and the European Union (European Social Fund-ESF) through the Operational Program “Human Resources Development, Education and Lifelong Learning 2014-2020” in the context of the project “The biomolecules of saliva and gingival cervical fluid as indicators of skeletal maturation in children” (MIS 5047928).