A new method for the separation and purification of the osteogenic compounds of nacre Ethanol Soluble Matrix

Nacre is able to induce bone-forming cells mineralization, and gains widely interest in bone regeneration. While, the osteoinductive compounds are not yet identified. ESM (Ethanol Soluble Matrix), a nacre extract from powder of Pinctada margaritifera pearl oyster shell, has been firstly proven having the capacity to induce mineralization and to restore mineralization defect in vitro. It is suitable to treat ESM as a source of osteoinductive compounds. Herein, we develop a new method for separating and purifying nacre extracts by an ionic approach. At first, cationic ESM (ESMc) and anionic ESM (ESMa) were achieved with ion-exchange resin. Then, ESM was separated and collected on cation exchange HPLC. Scanning Electron Microscopy coupled with Energy Dispersive X-ray Spectrometry (EDS) was used to reveal the concentrated elements in ESM fractions. A coupled cell models were used to test the ESM fractions. Alizarin Red staining was performed and quantified to evaluate the mineralization level. ESMc and 2 HPLC fractions stimulated the mineralization in both cells. EDS demonstrated the abundant presence of calcium and chloride in the osteogenic fractions. To validate, pure CaCl₂ was tested and proven having an osteogenic effect in both cells, but less stable than ESM. The mineralization nodules induced by ESM fractions and CaCl₂ differed in both cells. In conclusion, a new method was developed for separating and purifying nacre extracts by an ionic approach. By which, the osteoinductive compounds in ESM were proven cationic, and calcium in ESM was demonstrated to play a role in inducing the cell mineralization.