There is increasing evidence that cells of the epithelial root sheath synthesize enamel matrix proteins and that these proteins play a fundamental role in the formation of acellular cementum, the key tissue in the development of a functional periodontium. The purpose of the present study was to explore the effect of locally applied enamel matrix and different protein fractions of the matrix on periodontal regeneration in a buccal dehiscence model in monkeys. Buccal, mucoperiosteal flaps were raised from the canine to the 1st molar on each side of the maxilla. The buccal alveolar bone plate, the exposed periodontal ligament and cementum were removed. Various preparations of porcine enamel matrix with or without vehicles were applied before the flaps were repositioned and sutured. After 8 weeks, the healing was evaluated in the light microscope, and morphometric comparisons were made. Application of homogenized enamel matrix or an acidic extract of the matrix containing the hydrophobic, low molecular weight proteins, amelogenins, resulted in an almost complete regeneration of acellular cementum, firmly attached to the dentin and with collagenous fibers extending over to newly formed alveolar bone. After application of fractions obtained by neutral EDTA extraction containing the acidic, high molecular weight proteins of the enamel matrix, very little new cementum was formed and hardly any new bone. The results of the controls in which no test substance was applied before the repositioning of the flap, were very similar to those obtained with the EDTA extracted material. Propylene glycol alginate (PGA), hydroxyethyl cellulose and dextran were tried as vehicles for the enamel matrix preparations. Only PGA in combination with the amelogenin fraction resulted in significant regeneration of the periodontal tissues.

The recognition that periodontal regeneration can be achieved has resulted in increased efforts focused on understanding the mechanisms and factors required for restoring periodontal tissues so that clinical outcomes of such therapies are more predictable than those currently being used. In vitro models provide an excellent procedure for providing clues as to the mechanisms that may be required for regeneration of tissues. The investigations here were targeted at determining the ability of enamel matrix derivative (EMD) to influence specific properties of periodontal ligament cells in vitro. Properties of cells examined included migration, attachment, proliferation, biosynthetic activity and mineral nodule formation. Immunoassays were done to determine whether or not EMD retained known polypeptide factors. Results demonstrated that EMD under in vitro conditions formed protein aggregates, thereby providing a unique environment for cell-matrix interaction. Under these conditions, EMD: (a) enhanced proliferation of PDL cells, but not of epithelial cells; (b) increased total protein production by PDL cells; (c) promoted mineral nodule formation of PDL cells, as assayed by von Kossa staining; (d) had no significant effect on migration or attachment and spreading of cells within the limits of the assay systems used here. Next, EMD was screened for possible presence of specific molecules including: GM-CSF, calbindin D, EGF, fibronectin, bFGF, gamma-interferon, IL-1 beta, 2, 3, 6; IGF-1,2; NGF, PDGF, TNF, TGF beta. With immunoassays used, none of these molecules were identified in EMD. These in vitro studies support the concept that EMD can act as a positive matrix for cells at a regenerative site.

Cultured PDL cells exposed to EMD increase attachment rate, growth rate and metabolism, and subsequently release several growth factors into the medium. The cellular interaction with EMD generates an intracellular cAMP signal, after which cells secrete TGF-beta1, IL-6 and PDGF AB. Epithelial cell growth however, is inhibited by the same signal. This suggest that EMD favours mesenchymal cell growth over epithelium, and that autocrine growth factors released by PDL cells exposed to EMD contribute to periodontal healing and regeneration in a process mimicking natural root development.

Enamel Matrix Derivative (EMD) contains a protein complex belonging to the amelogenin family. Enamel matrix as well as EMD have been found to promote periodontal regeneration when applied onto denuded root surfaces in dehiscence models. In the present studies it is shown that propylene glycol alginate (PGA) is a suitable vehicle for EMD for its local application. EMD can be dissolved in PGA at an acidic pH, resulting in a highly viscous solution. At neutral pH and body temperature the viscosity decreases and EMD precipitates. Multilayers of EMD on mineral or protein surfaces have been analysed using ellipsometry, total internal reflection fluorescence (TIRF) and biospecific interaction analysis (BIA). The studies show that EMD adsorbs both to hydroxyapatite and collagen and to denuded dental roots. It forms insoluble spherical complexes, and detectable amounts remain at the site of application on the root surface for two weeks, as shown with radiolabelled protein in rats and pigs. Scanning electron micrograph (SEM) studies on monkey teeth further indicate that EMD in PGA may promote repopulation of fibroblast-like cells during the first weeks after application.

The aim of the present clinical trial was to test tolerability during 2 treatments with EMDOGAIN in a large number of patients. An open, controlled study design in 10 Swedish specialist clinics was chosen, with a test group of 107 patients treated with EMDOGAIN in connection with periodontal surgery at 2 surgical test sites per patient. The procedures were performed 2 to 6 weeks apart on one-rooted teeth with at least 4 mm deep intraosseous lesions. A control group of 33 patients underwent flap surgery without EMDOGAIN at 1 comparable site. In total, 214 test and 33 control surgeries were performed. Serum samples were obtained from test patients for analysis of total and specific antibody levels. 10 of the patients had samples taken before and after the first surgery, 56 other samples were taken after one treatment with EMDOGAIN, and 63 after 2 treatments. None of the samples, not even from allergy-prone patients after 2 treatments, indicated deviations from established baseline ranges. This indicates that the immunogenic potential of EMDOGAIN is extremely low when applied in conjunction with periodontal surgery. Comparison between the test and control groups demonstrated the same type and frequency of postsurgical experiences, i.e., reactions caused by the surgical procedure itself. Clinical probing and radiographic evaluation was performed at baseline and 8 months postsurgery. About half of the patients (44 test and 21 control) were also evaluated after 3 years. There was a significant difference between the test and control results at 8 months postsurgery, and this difference had increased further at the 3 year follow-up. The 2.5-3 mm increase in attachment and bone level after treatment with EMDOGAIN was of the same magnitude as seen in the studies with split-mouth design aiming for test of effectiveness of EMDOGAIN.