Purification and analysis of a 5kDa component of enamel matrix derivative.

High performance liquid chromatography (HPLC) methods were used to analyse a 5kDa component purified from enamel matrix derivative (EMD), the active ingredient in Emdogain, a commercial product for periodontal tissue regeneration. After initial purification by size-exclusion chromatography (SEC) on a 100 cm x 5 cm column (Bio-Gel P-30 Fine, 280 nm), collected fractions were analysed by size-exclusion HPLC (SE HPLC; TSK-Gel Super SW2000, 220 nm). The fractions containing only the 5kDa component were analysed by reversed-phase high-pressure chromatography (RP HPLC; YMC-Pack ODS-A, 200 nm), revealing four peaks of the 5kDa component. From 1200 mg of EMD (of which 9% is the 5kDa component), approximately 65 mg of lyophilised 5kDa component were obtained, corresponding to a recovery of 60%. The SE HPLC method was mainly suitable for qualitative analysis, whereas the RP HPLC method was appropriate for both qualitative and quantitative analysis.

Presence of an enamel matrix protein derivative on human teeth following periodontal surgery.

An enamel matrix protein derivative (EMD) has been successfully employed to restore fully functional periodontal ligament, cementum, and alveolar bone in patients with severe attachment loss. When applied to denuded root surfaces, EMD is believed to assemble into a matrix that locally stimulates regenerative responses in the surrounding tissues. Since wound healing and induction of regeneration are biological processes typically running from weeks to months after treatment, the EMD matrix must be present on the root surface over a biologically significant time period to be effective. So far, no studies have been done in humans on the persistence of EMD on the treated root surface. The aim of the present study was to investigate the persistence of the EMD-based matrix on periodontally involved human teeth treated surgically with EMD. Sixteen patients with one intrabony defect each around teeth scheduled for extraction due to advanced periodontitis were treated by means of access flap surgery with and without application of EMD. At 1, 2, 3, and 4 weeks postsurgery, the teeth were removed and processed for histology and immunohistochemistry. The slides were stained using a rabbit anti-enamel-matrix-derivative (anti-EMD) antibody and goat-antirabbit secondary antibodies labeled with horseradish peroxidase (HRP). Immunohistochemical evaluation demonstrated the presence of EMD on all test root surfaces during the entire observation period of 4 weeks. No EMD was detected on any of the control roots. The results demonstrate for the first time in humans that EMD is present on treated root surfaces for up to 4 weeks following periodontal surgery.