Cellular uptake and processing of enamel matrix derivative by human periodontal ligament fibroblasts

Abstract

Objective: Enamel matrix derivative (EMD), is an extract of porcine developing enamel matrix. Its commercialised form Emdogain, is claimed to stimulate periodontal regeneration by recapitulating original developmental processes, although the mechanism remains unclear. Our objective was to investigate interactions between EMD and human periodontal ligament (HPDL) fibroblasts in vitro.

Design: HPDL fibroblasts were cultured in the presence of fluorescently labelled EMD and cellular EMD uptake was monitored using confocal laser scanning microscopy and immunohistochemistry. Internalised EMD proteins were characterised using SDS-PAGE.

Results: EMD was internalised by HPDL fibroblasts leading to the appearance of multiple, vesicle-like structure in the cytoplasm. The internalised protein was composed mainly of the major 20kDa amelogenin component of EMD which was subsequently processed with time to generate a cumulative 5kDa component.

Conclusions: Cellular uptake and subsequent intracellular processing of EMD components by dental mesenchymal cells may play a role in EMD bioactivity and in part explain the turnover of Emdogain when placed clinically.

Fig. 1

Periodontal fibroblasts treated with EMD–FITC and viewed by confocal laser scanning microscopy. A typical image of confluent HPDL fibroblasts incubated in culture for 17 h with 0.5 mg/ml EMD–FITC and viewed in monolayer by confocal laser scanning microscopy. Multiple, strongly fluorescent vesicle like structures (VLSs) were observed within the cytoplasm of the cells. Some vesicles exhibited a dark non florescent area surrounding a fluorescent central region.

Fig. 2

Paraffin sections of EMD–FITC treated HPDL fibroblasts probed with anti-20 kDa-amelogenin antibodies. Cells were counterstained with haematoxylin and eosin. Multiple, strongly cross-reactive VLSs were evident within the cytoplasm (arrowed). Inset shows negative control (no primary antibody). No significant cross-reactivity observed.

Fig. 3

SDS-PAGE of whole EMD–FITC (as applied to the cells) and lysates of cells exposed to EMD–FITC for 1–17 h (viewed by UV transillumination). The composition of the intracellular material recovered after 1 h incubation with EMD–FITC conjugate (lane 2) reflected the composition of the applied EMD–FITC (lane 1) with the 20 kDa band being most prominent. Over 17 h there was a gradual accumulation of proteins, especially the 5 kDa protein which accrued to become the dominant band present at later time points (lanes 3–5).

Fig. 4

(a) SDS-PAGE of retrieved intracellular protein following incubation of the cells with EMD–FITC containing no 5 kDa protein. Although no 5 kDa protein was applied to the cells, material at this molecular weight still accumulated with time presumably due to intracellular degradation of a larger precursor (e.g. the 20 kDa protein). (b) SDS-PAGE of retrieved intracellular proteins following incubation of the cells with FITC labelled 5 kDa EMD fraction. No uptake was detected.