Loss of attachment in the marginal periodontium of the rat incisor under non-inflammatory conditions. Expression of alkaline phosphatase activity. Experimental Oral Biology Group.

Alkaline phosphatase (ALP) has been suggested to play a role in acellular cementum formation and maintenance of periodontal attachment. In an attempt to determine whether changes in attachment level are associated with altered expression of ALP-activity in the periodontium we induced natural loss of attachment in rats by pinning the lower incisor to the jaw bone. Previous studies have shown that this procedure results in regressive changes in the marginal periodontium without any inflammatory response. Six months after blockage of eruption the attachment level on the experimental (right) side had shifted about 700 microm in the apical direction. On the control (left) side the apical termination of the junctional epithelium had remained stationary with respect to the alveolar crest. Our observations have shown that during the first few weeks of the experiment loss of attachment is accompanied by considerable reduction of ALP-activity in the supracrestal part of the periodontium. At later time intervals, however, no distinct relation was found between apical migration of junctional epithelium and loss of ALP-activity in the supracrestal region, indicating that the two phenomena are not directly related to each other. The domain of the ALP-positive fibroblasts in the supracrestal extension of the periodontal ligament decreased in size and was replaced by ALP-negative connective tissue cells probably coming from the outer gingival domain. Since at all time intervals a distinct demarcation could be observed between the ALP-positive and ALP-negative areas, we interpret our data as indicating that ligament and gingival cells do not mix.

Cell-bound and extracellular matrix-associated alkaline phosphatase activity in rat periodontal ligament. Experimental Oral Biology Group.

In previous studies it was noted that alkaline phosphatase (ALP) activity in periodontal ligament does not only seem to be related to cells but may also be associated with the extracellular matrix. In an attempt to clarify this we studied the distribution of the enzyme at the electron microscopic level. In addition, ALP-activity was assessed biochemically following extraction of the ligament with (i) agents dissolving the membrane or splitting the phosphatidylinositol anchor (Triton X-100 or phosphatidylinositol-phospholipase C, respectively), and (ii) a matrix-degrading enzyme cocktail (collagenase, hyaluronidase and elastase). Histochemical observations revealed (a) a heterogeneous distribution of ALP-activity, with highest activity adjacent to the alveolar bone and (b) two pools of activity; one bound to cells and one associated with the collagenous extracellular matrix. In line with this were the biochemical data indicating that approximately 10% of the enzyme activity was firmly bound to the extracellular matrix and 90% to plasma membranes. Isoelectric focusing did not reveal differences between the two fractions, both samples yielding a single broad band corresponding with an isoelectric point of about 4.4.

Alkaline phosphatase activity in the periodontal ligament and gingiva of the rat molar: its relation to cementum formation.

Alkaline phosphatase (ALP) is a glycoprotein thought to be involved in processes leading to mineral formation in tissues like bone and cementum. In the rat molar periodontium, several regions are associated with the formation of cementum (periodontal ligament, inner part of the gingiva), whereas other areas are not (e.g., the outer part of the gingiva just beneath the outer oral epithelium). In an attempt to establish how the spatial distribution of ALP activity relates to cementum formation, we assessed the activity of the enzyme quantitatively in the periodontium of the rat maxillary molars, by using the indoxyl-tetrazolium salt method. It appeared that the distribution of enzyme activity in the ligament was heterogeneous, indicating local variations in the phosphate household. Highest activity was found in areas related to mineralization, adjacent to the alveolar bone and cementum. Enzyme activity was higher adjacent to cellular cementum than to acellular cementum. With respect to acellular cementum, a highly significant positive correlation was found between ALP activity and cementum thickness, which indicates a close relationship between local phosphate production and cementum formation rate. An interesting observation in the connective tissue of the gingiva mesial to the first molar was a sharp demarcation between an ALP-positive inner part, adjacent to the tooth, and an ALP-negative outer part, underneath the outer oral epithelium. In the interdental gingiva, the entire connective tissue proved positive for the enzyme, suggesting that this region consists of the combined inner gingival parts of two adjacent teeth.

Formation of afibrillar acellular cementum-like layers induced by alkaline phosphatase activity from periodontal ligament explants maintained in vitro.

Fibroblasts of the periodontal ligament, by their alkaline phosphatase (ALP) activity, are considered to play a role in the formation of acellular cementum. As a means of exploring this hypothesis, periodontal ligament explants from rat incisors were cultured in direct contact with bovine dentin slices in the presence of 10 mmol/L beta-glycerophosphate. Periosteal and pericardial tissue explants were maintained under similar conditions. After two weeks, the slices were harvested and processed for electron microscopic examination. Controls included periodontal ligament explants to which the ALP-inhibitor levamisole was added. The results suggest that only ALP-positive cultures from periodontal ligament and periosteum form mineralized layers along the dentin. After demineralization, layers consisted of fine filamentous or granular material of moderate electron-density and resembled afibrillar acellular cementum. Our findings support the hypothesis that periodontal ligament fibroblasts, by means of their ALP activity, play a pivotal role in the formation of acellular cementum.

A quantitative enzyme histochemical analysis of the distribution of alkaline phosphatase activity in the periodontal ligament of the rat incisor.

The spatial distribution of alkaline phosphatase (ALP) activity was examined in the periodontal ligament of the continuously growing rat incisor. With the indoxyl-tetrazolium salt method, enzyme activity was demonstrated in undecalcified cryosections, and the amount of reaction product was quantified. ALP activity appeared to be distributed heterogeneously. Its highest activity was found in the bone-related compartment of the ligament. In the tooth-related compartment and the supracrestal extension of the ligament, enzyme activity was significantly lower, but still higher than in the lamina propria of the gingiva. In the part of the ligament bordering the cementum, highest activity was found in the apical region just occlusal to Hertwig's epithelial root sheath, where formation of acellular cementum begins. From there toward the incisal edge, the activity of the enzyme gradually decreased. It is suggested that differences among the various parts of the periodontal ligament are related to local variations in phosphate metabolism and cementum deposition.