Enamel matrix proteins; old molecules for new applications

Abstract

Emdogain (enamel matrix derivative, EMD) is well recognized in periodontology, where it is used as a local adjunct to periodontal surgery to stimulate regeneration of periodontal tissues lost to periodontal disease. The biological effect of EMD is through stimulation of local growth factor secretion and cytokine expression in the treated tissues, inducing a regenerative process that mimics odontogenesis. The major (>95%) component of EMD is Amelogenins (Amel). No other active components have so far been isolated from EMD, and several studies have shown that purified amelogenins can induce the same effect as the complete EMD. Amelogenins comprise a family of highly conserved extracellular matrix proteins derived from one gene. Amelogenin structure and function is evolutionary well conserved, suggesting a profound role in biomineralization and hard tissue formation. A special feature of amelogenins is that under physiological conditions the proteins self-assembles into nanospheres that constitute an extracellular matrix. In the body, this matrix is slowly digested by specific extracellular proteolytic enzymes (matrix metalloproteinase) in a controlled process, releasing bioactive peptides to the surrounding tissues for weeks after application. Based on clinical and experimental observations in periodontology indicating that amelogenins can have a significant positive influence on wound healing, bone formation and root resorption, several new applications for amelogenins have been suggested. New experiments now confirm that amelogenins have potential for being used also in the fields of endodontics, bone regeneration, implantology, traumatology, and wound care.

Figures

Fig. 1

A schematic diagram of extracellular amelogenin proteolytic processing: After secretion from the cell the 20 kDa Amelogenin is processed into smaller peptides by specific proteases. Eventually, the smaller peptides become soluble and are released from the insoluble amelogenin assemblies. TRAP, tyrosine-rich amelogenin peptide, is one candidate for an active peptide that can interact with cellular receptors.

Fig. 2

Transmission electron micrographs (TEM) of amelogenin assemblies being transported over the cell membrane in clathrin-coated pits (CCP) in a primary human osteoblast. Osteoblasts were incubated with enamel matrix derivative (EMD; 50 μg/ml) for 3 h. EMD assemblies are stained with a gold-labeled anti-amelogenin antibody (black dots). The CCP is in center of the circle. Further analysis have showed that once in the ccp the EMD assemblies colocalize with the clathrin adaptor–protein complex, AP-2, the major mechanism of cargo sorting into coated pits in mammalian cells, suggesting that uptake of amelogenin is an active process involving specific receptors. Scale bar = 400 nm.

Fig. 3

Longitudinal sections through pig incisors 4 weeks after pulpotomy and treatment with (A) calcium hydroxide and (B) enamel matrix derivative (EMD). Formation of new dentin (ND) completely bridging the defect is clearly visible in the EMD-treated tooth, whereas only partial closing of the defect is seen in the calcium hydroxide-treated tooth. C is cavity, PD is primary dentin and P is pulp. Scale bar = 1.0 mm.

Fig. 4

Schematic drawing of the transforming growth factor (TGF)-β pathway: enamel matrix derivative activation of the TGF-β pathway in osteoblasts is clearly visualized by up-regulation of the Smad genes (green) in the gene array studies, generating a nuclear signal for new gene expression. Green symbols indicate a more than fourfold up-regulation of expression of the actual gene. Red indicates a fourfold or more down-regulation. Gray symbols are genes regulated more than twofold (up or down). This drawing is based on data from gene array studies performed on primary human osteoblasts stimulated with amelogenins for 24 h.

Fig. 5

Primary wound healing in full thickness wounds in pigs after treatment with enamel matrix derivative (EMD) or control (propylene glycol alginate vehicle only). After 3 days the EMD-treated wound is significantly better vascularized than the control as visualized here by the vivid red color and presence of blood vessels in the wound surface. After 11 days the EMD-treated wound is almost completely closed and epithelium is covering most of the wound. At this stage the control wound is still covered in granulation tissue and epithelialization has not yet started. After 15 days the EMD-treated wound is completely covered by epithelium, and most of the wound cavity is filled in, while the control wound still show remnants of granulation tissue exposed in the middle of the wound. In this study EMD-treated wounds healed twice as fast as control wounds (the vehicle control used here, polyglycol alginate, is often used in wound care because of its excellent biocompatibility and low pH that restrict bacterial growth and aid healing).