Studies during the last 20 years have indicated that enamel-related proteins are involved in the formation of cementum. In the present article, this relation is further explored. Attention is called to the fact that coronal acellular extrinsic fiber cementum is formed on the enamel surface in a number of species. The composition of the enamel matrix proteins and the expression of these proteins during root formation are briefly reviewed. The dominating constituent of the enamel matrix, amelogenin, is shown by means of immunohistochemistry to be expressed in human teeth during root formation. Amelogenin was also found to be present in Tomes' granular layer of human teeth. When mesenchymal cells of the dental follicle were exposed to the enamel matrix a non-cellular hard tissue matrix was formed at the enamel surface. Application of porcine enamel matrix in experimental cavities in the roots of incisors of monkeys induced formation of acellular cementum that was well attached to the dentin. In control cavities without enamel matrix, a cellular, poorly attached hard tissue was formed. The present studies provide additional support to the idea that enamel matrix proteins are involved in the formation of acellular cementum and also that they have the potential to induce regeneration of the same type of cementum.

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.

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.

SUMMARYMineralized tissues are unique in using proteins to attract and organize calcium and phosphate ions into a structured mineral phase. A precise knowledge of the expression and extracellular distribution of matrix proteins is therefore very important in understanding their function. The purpose of this investigation was to obtain comparative information on the expression, intracellular and extracellular distribution, and dynamics of proteins representative of the two main classes of enamel matrix proteins. Amelogenins were visualized using an antibody and an mRNA probe prepared against the major alternatively spliced isoform in rodents, and nonamelogenins by antibodies and mRNA probes specific to one enamel protein referred to by three names: ameloblastin, amelin, and sheathlin. Qualitative and quantitative immunocytochemistry, in combination with immunoblotting and in situ hybridization, indicated a correlation between mRNA signal and sites of protein secretion for amelogenin, but not for ameloblastin, during the early presecretory and midto late maturation stages, during which mRNA signals were detected but no proteins appeared to be secreted. Extracellular amelogenin immunoreactivity was generally weak near secretory surfaces, increasing over a distance of about 1.25 m to reach a level slightly above an amount expected if the protein were being deposited evenly across the enamel layer. Immunolabeling for ameloblastin showed an inverse pattern, with relatively more gold particles near secretory surfaces and much fewer deeper into the enamel layer. Administration of brefeldin A and cycloheximide to stop protein secretion revealed that the immunoblotting pattern of amelogenin was relatively stable, whereas ameloblastin broke down rapidly into lower molecular weight fragments. The distance from the cell surface at which immunolabeling for amelogenin stabilized generally corresponded to the point at which that for ameloblastin started to show a net reduction. These data suggest a correlation between the distribution of amelogenin and ameloblastin and that intact ameloblastin has a transient role in promoting/stabilizing crystal elongation. A meloblasts , like all hard tissue-forming cells, release an intricate set of extracellular matrix proteins optimized for promoting the development of a closely associated mineral phase (reviewed in Deutsch et al.

The present experimental studies in monkeys were undertaken to study the initiation and progression of dentoalveolar ankylosis of replanted teeth and associated root resorption. Maxillary and mandibular lateral incisors were extracted and replanted after an extraoral period of 15 min or 1 h. Teeth with an extraoral period of 1 h were endodontically treated. Half the number of monkeys were given antibiotics at the time of replantation. The observation periods varied from 2 days to 40 weeks. Irrespective of the length of the extraoral period, initial root resorption and minor areas of ankylosis were found 1 week after replantation. The initial ankylosis was not preceded by root resorption. In teeth replanted after an extraoral period of 15 min the ankylotic area did not increase with increasing time after replantation. Instead the periodontal membrane was re-established, separating the root surface from the alveolar bone. In teeth replanted after an extraoral period of 1 h, the initial ankylotic area increased with increasing time after replantation. Eight weeks and more after replantation, most of the periodontal membrane was replaced by bone covered by osteoblasts and occasional osteoblasts that were in continuity with the endosteal cells outlining the marrow spaces of the alveolar bone. The cementum and dentin were then gradually resorbed with increasing time after replantation. Antibiotics given at the time of replantation reduced the initial inflammation in the periodontal membrane and the inflammatory root resorption after all observation periods and it also seemed to some extent to prevent bacteria from entering the necrotic pulp tissue. Based on the present results it is suggested that root resorption associated with dentoalveolar ankylosis is initiated by endosteal osteoblasts and is thus a hormonally regulated process. This is in contrast to inflammatory root resorption, which seems to be triggered by inflammatory cells.

Administration of antibiotics is usually recommended when a traumatieally avulsed tooth is replanted, in order to prevent bacterial contamination. In the present study, permanent lateral incisors of monkeys were extracted, allowed to dry for 1 h and then replanted. Some teeth had their pulp chambers opened labially and left open for 3 wk after replantation. The monkeys were treated with antibiotics (i.m.) eitlicr at the time of replantation or 3 wk after replantation. In some monkeys, antibiotics were placed in the pulpal cavity. Cotnparisons were made of the effect of endodontic treatment on periodontal healing and root resorption. The tnonkeys were killed 8 wk alter replantation. It was found that after systemic antibiotic treatment at the time of replantation there was no inflammatory root resorption. Also, endodontic treatment at the time oi replantation prevented inflammatory root resorption. When systemic antibiotic treatment was instituted 3 wk after the replantation, there was no reduction of the inflammatory root resorption as compared with teeth without antibiotic treatment. Application of antibiotics in the pulp 3 wk after replantation almost completely eliminated the inflammatory resorption. In all the treatment groups where administration of antibiotics or endodontic treatment had prevented, reduced or eliminated inflammatory root resorption, 30-45% of the root surface area was ankylotic 8 wk after replantation.

Extracted monkey teeth were endodontically treated, stored in milk or saliva for two or six h, and then replanted. Periodontal conditions were evaluated after eight wk. Teeth that had been stored for two or six h in milk or for two h in saliva showed periodontal healing almost as good as that of immediately replanted teeth. Teeth that had been kept in saliva for six h or bench-dried for one h showed extensive replacement resorption. Milk may thus be recommended as a storage medium for ex-articulated teeth prior to replantation in cases when immediate replantation is not possible.