Formation Of Acellular Cementum Research Articles

Treatment of intrabony periodontal defects with enamel matrix derivative: a literature review.

The enamel matrix derivative (EMD) has been recently introduced in the periodontal field to overcome short-comings associated with currently available regenerative techniques. Information accumulated over the past years with application of EMD guided regeneration (EGR) in intrabony periodontal defects allowed a thorough evidence-based retrospective analysis. Clinical data from EMD controlled studies were pooled for meta-analysis and weighted according to the number of treated defects. Clinical attachment gain amounted to 3.2 +/- 0.9 mm (33% of the original attachment level) and probing reduction averaged 4.0 +/- 0.9 mm (50% of the baseline probing depth) for a total of 317 lesions with a mean baseline depth of 5.4 +/- 0.8 mm. Improvements in clinical parameters achieved with EMD were statistically significant in reference to preoperative measurements. However, despite the overall efficacy of EGR therapy, a significant variation in clinical outcomes was observed. Similar therapeutic results were reported in studies where EGR was compared directly to guided tissue regeneration. However, the controlled clinical trials did not have adequate statistical power to firmly support superiority or equivalency between the 2 regenerative therapies. The statistical superiority of EGR over treatment with open flap debridement has been established. Preliminary histologic investigations with surgically created defects and experimental periodontal lesions demonstrated the ability of EGR to induce formation of acellular cementum and promote significant anaplasis of the supporting periodontal tissues. The potential of EMD to encourage periodontal regeneration was also confirmed in human intrabony defects. However, recent human histologic studies have questioned both the consistency of the histologic outcomes and the ability of EGR to predictably stimulate formation of acellular cementum. Identifying clinical modifying parameters and understanding cellular interactions are apparently essential for the development of methodologies to enhance predictability and extent of EGR clinical and histologic results.

Distinctive expression of extracellular matrix molecules at mRNA and protein levels during formation of cellular and acellular cementum in the rat.

Little is known about differential expression of extracellular matrices secreted by cementoblasts between cellular and acellular cementum. We hypothesize that cementoblasts lining acellular cementum express extracellular matrix genes differently from those lining cellular cementum, thereby forming two distinct types of extracellular matrices. To test this hypothesis, we investigated spatial and temporal gene expression of selected extracellular matrix molecules, that is type I collagen, bone sialoprotein, osteocalcin and osteopontin, during formation of both cellular and acellular cementum using in situ hybridization. In addition, their extracellularly deposited and accumulated proteins were examined immunohistochemically. The mRNA transcripts of pro-alpha1 (I) collagen were primarily localized in cementoblasts of cellular cementum and cementocytes, while those of bone sialoprotein were predominantly seen in cementoblasts lining acellular cementum. In contrast, osteocalcin was expressed by both types of cementoblasts and cementocytes and so was osteopontin but only transiently. Our immunohistochemical examination revealed that translated proteins were localized extracellularly where the genes had been expressed intracellularly. The present study demonstrated the distinctive expression of genes and proteins of the extracellular matrix molecules between cellular and acellular cementum.

Porcine fetal enamel matrix derivative enhances bone formation induced by demineralized freeze dried bone allograft in vivo.

Embryonic enamel matrix proteins are involved in the formation of acellular cementum during development of the periodontal attachment apparatus, suggesting that these proteins might be used clinically to promote periodontal regeneration. At present, it is unknown if these proteins are osteoinductive, osteoconductive, or osteopromotive. To address this question, we examined the ability of a commercially prepared embryonic porcine enamel matrix derivative to induce new bone formation in nude mouse calf muscle, or to enhance the bone induction ability of a demineralized freeze-dried bone allograft (DFDBA). Porcine fetal enamel matrix derivative (EMD) was implanted bilaterally in the calf muscle of 4 male Nu/Nu mice per treatment group (N = 8 implants): 2 mg EMD alone; 4 mg EMD alone; inactive human DFDBA alone; inactive DFDBA + 2 mg EMD; inactive DFDBA + 4 mg EMD; active DFDBA alone; active DFDBA + 2 mg EMD; and active DFDBA + 4 mg EMD. Implants were harvested after 56 days and examined histologically for bone induction using a semi-quantitative score and histomorphometrically for area of new bone, cortical bone, bone marrow, and residual DFDBA. Implants containing inactive DFDBA, 2 mg EMD, 4 mg EMD, and inactive DFDBA + 2 or 4 mg EMD did not induce new bone. Active DFDBA and active DFDBA + 2 mg EMD induced new bone to a similar extent. In contrast, active DFDBA + 4 mg EMD resulted in enhanced bone induction, area of new bone, and cortical bone. Residual DFDBA was also increased in this group. EMD is not osteoinductive. However, it is osteopromotive, due in part to its osteoconductive properties, but a threshold concentration is required.

Porcine fetal enamel matrix derivative stimulates proliferation but not differentiation of pre-osteoblastic 2T9 cells, inhibits proliferation and stimulates differentiation of osteoblast-like MG63 cells, and increases proliferation and differentiation of normal human osteoblast NHOst cells.

Embryonic enamel matrix proteins are hypothesized to be involved in the formation of acellular cementum during tooth development, suggesting that these proteins can be used to regenerate periodontal tissues. Enamel matrix protein derived from embryonic porcine tooth germs is used clinically, but the mechanisms by which it promotes the formation of cementum, periodontal ligament, and bone are not well understood. This study examined the response of osteoblasts at 3 stages of osteogenic maturation to porcine fetal enamel matrix derivative (EMD). Proliferation (cell number and [3H]-thymidine incorporation), differentiation (alkaline phosphatase and osteocalcin), matrix synthesis ([35S]-sulfate incorporation; percentage of collagen production), and local factor production (prostaglandin E2 [PGE2] and transforming growth factor-beta 1 [TGF-beta1]) were measured in cultures of 2T9 cells (pre-osteoblasts which exhibit osteogenesis in response to bone morphogenetic protein-2 [BMP-2]), MG63 human osteoblast-like osteosarcoma cells, and normal human osteoblasts (NHOst cells). EMD regulated osteoblast proliferation and differentiation, but the effects were cell-specific. In 2T9 cell cultures, EMD increased proliferation but had no effect on alkaline phosphatase-specific activity. EMD decreased proliferation of MG63 cells and increased cellular alkaline phosphatase and osteocalcin production. There was no effect on collagen synthesis, proteoglycan sulfation, or PGE2 production; however, TGF-beta1 content of the conditioned media was increased. There was a 60-fold increase in cell number in third passage NHOst cells cultured for 35 days in the presence of EMD. EMD also caused a biphasic increase in alkaline phosphatase that was maximal at day 14. EMD affects early states of osteoblastic maturation by stimulating proliferation, but as cells mature in the lineage, EMD enhances differentiation.

Epithelial Dlx-2 homeogene expression and cementogenesis.

The Dlx-2 (distal-less gene) homeoprotein transcription factor controls early tooth development but has not been studied during the late stages of biomineralization. Transgenic mice containing a Dlx-2/LacZ reporter construct were used to map the Dlx-2 expression pattern in cementoblasts, the dental cells most closely related to bone cells and therefore suggested to be uniquely positioned osteoblasts. During initial root formation, marked expression of Dlx-2 was evident in molar and incisor root epithelium, whereas dental papilla and follicle were negative. Dlx-2 was expressed in this epithelium from the apical loop to the area of its disruption. During acellular cementum formation in both incisors and molars, Dlx-2 expression was observed in the majority of differentiated cementoblasts from the apical region to the erupting zones. During cellular cementum formation, the presence of which characterizes growth-limited molars, Dlx-2 expression was restricted to the innermost cementoblasts and entrapped cementocytes. These data further support the hypothesis of a complex origin and fate of cementum-forming cells, as previously suggested by the expression patterns of a set of mesenchymal and epithelial markers, notably ameloblastin as shown here. Dlx-2 expression might constitute a landmark of cementoblast subpopulations of epithelial origin. (J Histochem Cytochem 48:277-283, 2000)

Root development in mice lacking functional tissue non-specific alkaline phosphatase gene: inhibition of acellular cementum formation.

Tissue non-specific alkaline phosphatase (TNAP) is richly present in developing teeth including the cells of the periodontal ligament. Here, we investigated tooth and root development in mice lacking the TNAP gene. Heterozygous mutants were obtained from The Jackson Laboratory, Animal Resources (Bar Harbor, ME, USA) and bred. TNAP-deficient mice and their littermates were killed from 6 to 25 days after birth and their molar blocks processed for light and electron microscopy. It was observed that the eruption of the incisors into the oral cavity was delayed for 2 to 3 days. Also, the onset of mineralization of the mantle dentin in the roots of the developing molars was delayed for 2 to 3 days. Yet, dentin and enamel formation in the homozygous mutants showed a more or less normal pattern, with the exception of localized enamel hypoplasias. The most conspicuous finding was the defective formation of acellular cementum along the molar roots. Instead of a continuous layer, the cementum was deposited as very thin and irregularly shaped patches around the bases of the periodontal ligament fibers. Sharpey's fibers were short and poorly developed. In contrast, the development of the alveolar bone, the periodontal ligament, and the cellular cementum was seemingly unaffected. It is concluded that TNAP represents an essential factor in mantle dentin mineralization and in the formation of acellular cementum.

Biological mediators for periodontal regeneration.

A review of the literature on the use of growth-regulatory molecules in the oral cavity permits a model in which to consider approaches to oral tissue engineering. These concepts apply to periodontal regeneration and to regeneration of alveolar bone. In either case, the formation of tissues is complex but proceeds in a deliberate and orderly sequence. In these sequence of events resulting in either bone or cementum formation, periodontal ligament and bone can be stimulated at various points. Different signals can apparently be used to stimulate tissue formation including mitogenic signals and differentiation factors. Additionally, both hard and soft tissue stimulatory molecules appear to be permissive. Classic receptor-mediated peptides or extracellular matrix molecules for soft and hard tissues appear to allow stimulation of tissue formation cascades. Importantly, it also appears that the stimulatory event is transitory (that is, short-lived) and leads itself to a sequence of cellular events. These cellular events in turn stimulate a number of subsequent events (such as chemotaxis, proliferation, differentiation or angiogenesis), which lead to further progression of tissue formation. While a solid scientific rationale exists for the use of a variety of growth and attachment factors in regeneration of oral tissues, only a small number are being pursued clinically. Many therapeutic regimens have failed in preclinical testing or have resulted in limited regenerative capacity. The mitogenic polypeptides that stimulate soft tissue growth (such as platelet-derived growth factor) and both hard and soft tissue growth (such as transforming growth factor-beta) appear to have not led to successful enough outcomes to facilitate further work towards regulatory approval. The demonstrated ability of bone morphogenetic proteins to generate substantial quantities of bone suggest many applications in the oral cavity where this is the only tissue desired. Another therapeutic candidate is enamel matrix derivative, a set of matrix proteins. Enamel matrix derivative appears to stimulate first acellular cementum formation, which may allow for functional periodontal ligament formation. It will be of interest in the future to determine whether the protein matrix contains classic mitogenic or differentiation factors as well as the amelogenins. It is also evident that the bone morphogenetic proteins permit periodontal ligament formation. The conditions for stimulating predictable periodontal ligament tissues with bone morphogenetic proteins however are not known. It is clear that the bone morphogenetic proteins are excellent molecules for stimulating oral bone formation. The results of all these studies will determine the future therapeutic potential for these growth molecules such that they may be used to optimally stimulate and direct specific points along tissue formation cascades.

The structure and function of periodontal ligament cells in acellular cementum in rat molars

To elucidate the structure and function of periodontal ligament cells at the periodontal ligament-cementum interface in advanced acellular cementogenesis, the cervical regions of molars in rats aged 6 weeks were observed by light and electron microscopy. The light and transmission electron microscopy showed the periodontal ligament cells to be elongated between dense, well-developed principal fibers. The transmission and scanning electron microscopy showed that these cells extended wing-like projections from the lateral surface, forming cylindrical compartments surrounding the principal fibers. In addition, finger-like projections extended toward the cementum from the cementum-facing ends. The main results suggest the following: at the periodontal ligament-cementum interface, the periodontal ligament cells maintain the architecture of the principal fibers by means of extracellular compartments. The arrangement of finger-like projections results in the formation of acellular cementum containing only Sharpey's fibers as a fibrous component.

GTR treatment of degree III furcation defects following application of enamel matrix proteins. An experimental study in dogs.

The aim of the present study was to evaluate the effect of enamel matrix proteins (EMD) on periodontal wound healing in degree III furcation defects in dogs. The experiment was performed in 5 foxhound dogs. 2 months prior to the start of the experiment, the 2nd and 4th lower premolars were extracted. Degree III furcation defects were created in the 3rd mandibular premolars (3P3). The furcation defects were subsequently exposed to reconstructive surgery. Buccal and lingual full thickness flaps were elevated in the lower premolar regions. The exposed root surfaces of the experimental teeth were planed. A notch was placed in the roots at the base of the defect. In one side of the mandible (Test group), phosphoric acid gel was applied over the root surfaces for 15 s. The acid was removed by flushing the root surfaces with sterile saline. Subsequently, a gel of EMD was applied to cover all instrumented root surfaces. Following gel application, a resorbable barrier membrane was adjusted to cover the buccal and lingual entrances of the furcation defect. The flaps were repositioned to cover the barrier and sutured. The contralateral premolar (Control group) received the same treatment, but acid etching was not performed and EMD was not applied prior to barrier installation. 4 months after reconstructive surgery, the animals were sacrificed and biopsies from the 3P3 regions harvested. The biopsies were placed in a fixative, demineralized in EDTA, dehydrated and embedded in paraffin. 3 mesiodistal sections, representing the central portion of the furcation site, were selected for histological analysis of the defect. The furcation defects of both the Test and Control groups were clinically closed and were found to harbor bone and periodontal ligament tissue which appeared to be in structural continuity with a newly formed root cementum. The relative amounts of mineralized bone, bone marrow and periodontal ligament tissue that had formed were similar in the Test and the Control group. In the Test group, however, the cementum that had formed in the apical portion of the furcation defect was different from the corresponding tissue in the coronal portion, and also different from the cementum observed in the Control group. In the apical portion of the test defect a thin (12 microm) acellular cementum had been laid down, while in the coronal portion a thick (32 microm) cellular cementum, similar to the cementum found in the Control group, could be observed. The current observation, hence, seems to confirm that EMD when applied onto an instrumented and acid etched dentine surface may create an environment conducive for the formation of acellular cementum.

Formation of reparative acellular extrinsic fiber cementum in relation to implant materials installed in rat periodontium.

The aim of the present study was to determine the nature of the cells associated with the formation of reparative acellular extrinsic fiber cementum (AEFC). Calcifying collagen membranes, hydroxyapatite particles and/or non-resorbable ePTFE membranes were implanted in lesions made in the periodontium of the rat mandibular incisor. The incisor was prevented from erupting further, and the animals were killed after 1-8 wk. In the first week, next to cells with a fibroblastic phenotype, epithelial cells (ECs) migrating out of the reduced enamel epithelium were among the cells colonizing the wounds. From 2 wk on, the ECs showed regressive changes and disappeared. Along mineralized implant surfaces, a basophilic layer enriched in osteopontin (but without detectable amelogenin) was deposited. After 3 wk (when ECs were no longer present) the healing tissue transformed into a well-organized PDL-like tissue, and AEFC started to develop. Along the non-mineralizing ePTFE membranes, AEFC did not form except in regions where small calcified bodies were present. It is concluded that reparative AEFC layers are formed only along calcified surfaces. The cells associated with this reparative activity are periodontal ligament cells with a fibroblastic phenotype.

Epithelial Rests Colocalize with Cementoblasts Forming Acellular Cementum but Not with Cementoblasts Forming Cellular Cementum

Epithelial rests of Malassez and cementoblasts were examined in the rat molars during the early stages of root formation using an antilaminin antibody and/or peanut agglutinin (PNA), and an antiosteocalcin (OC) antibody, respectively. The roots of the first molars were used for study. The antilaminin antibody stained the basement membrane surrounding the epithelial root sheath and epithelial rests. The basement membrane of the epithelial root sheath was continuous, but that of the epithelial rests was discontinuous. The cells of epithelial rests and epithelial root sheath were positive for PNA. The structural characteristics of the epithelial rests were seen in the sections stained doubly with PNA and the antilaminin antibody. The cells of epithelial rests were fibroblast-like and formed a fine mesh in 2-week-old rats. In 3-week-old rats, the epithelial rests were also present at the coronal half of root surface, showing typical cell cords, but were not present at the apical part of the root surface where the cellular cementum covered the root dentin. At the root apex of 3-week-old rats, the cells of epithelial rests forming fine meshes were seen near the epithelial root sheath. The anti-OC antibody stained cementoblasts lining acellular and cellular cementum. The sections doubly stained with the anti-OC and the antilaminin antibodies or PNA further revealed the close relation between epithelial rests and cementoblasts. The OC-positive cells lining acellular cementum or dentin were localized very close to the epithelial rests. In contrast, the OC-positive cells lining cellular cementum did not show close association with the epithelial cells, except the cells located most apically where the basement membrane of the epithelial root sheath is disrupted and the initial cellular cementum begins to be formed. The present results suggest that the epithelial rests and/or the discontinuous basement membrane of them may have a role for the acellular cementum formation at least in the early stage of root formation.

Enamel matrix, cementum development and regeneration.

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.

Ultimate tensile strength of PDL of molars in rats after 1-hydroxyethylidene-1, 1-bisphosphonate injections

Objectives and methods: Administration during root formation of a bisphosphonate, 1-hydroxyethylidene-1, 1-bisphosphonate (HEBP), at a dose corresponding to 10mg P/kg body weight, has been found to interfere with the formation of acellular cementum in rats. The purpose of this study was to measure the force required to extract a tooth lacking normal acellular cementum, and to correlate this force and the ultimate periodontal strength with the morphology of the periodontal tissues at different time intervals after single or multiple injections of HEBP. Results: A single injection of HEBP given during root formation inhibited the formation of acellular cementum and resulted in a temporary reduction of the extraction force and the ultimate tensile strength. Ninety days after the injection of HEBP, both parameters were the same as in the controls. The increase in extraction force and ultimate tensile strength was associated with the onset of occlusal contact of the teeth. The organization of periodontal ligaments was improved after the teeth reached the occlusal level. After daily injections of HEBP for 3 days, there was a permanent reduction in root length and dento-alveolar ankylosis developed in the furcation area. Conclusion: (1) A single or three injections of HEBP changed the formation of acellular cementum to that of an atypical hyperplastic cementum which increased the resorption risk at this site. (2) The ultimate tensile strength was markedly reduced in teeth lacking acellular cementum.

The localization of epithelial root sheath cells during cementum formation in rat molars.

The purpose of this study was to investigate the distribution of epithelial cells and the fate of the basement membrane along the root surface of rat molars during cementogenesis, and to test the hypothesis that the Hertwig's epithelial root sheath (HERS) cells remain on the root surface if mineralization is inhibited. To demonstrate the HERS cells and basement membrane, immunohistochemistry with antibodies against keratin and laminin were used. The dentin matrix mineralization was inhibited by a single injection of 1-hydroxyethylidene-1, 1-bisphosphonate (HEBP). A modified Gomori staining method was used to monitor the inhibition of mineral formation in dentin and cementum. Paraffin sections were stained with haematoxylin-eosin, and freeze-dried sections were used for Gomori and immunohistochemical stainings. We found that the formation of acellular cementum was suppressed above the dentin with inhibited mineralization. Instead, a hyperplastic matrix, different from acellular cementum, covered the dentin. This hyperplastic cementum had keratin- and laminin-positive cells incorporated; such cells were never incorporated in normal acellular cementum. The later formation of cellular cementum correlated, in controls, with the disappearance of HERS cells from the root surface. Treatment with HEBP resulted in a persistent presence of epithelial cells, interpreted as an inhibition of their disappearance. In conclusion, there is evidence that the cells of HERS are involved in the development of both acellular and cellular cementum. The developmental processes of these tissues appear in some way to be influenced by or associated with the initial mineralization of the dentin.

Origins of cementum.

There are diverging opinions about the role of Hertwig's epithelial root sheath in the formation of cementum. In the present review this role is discussed. There is increasing evidence that Hertwig's epithelial root sheath is actively involved in the formation of both acellular and cellular cementum. The development of acellular cementum seems to be associated with secretion of enamel-related proteins by cells of the epithelial root sheath. Formation of the matrix for cellular cementum appears to be induced by exposure of the inner layer of the epithelial root sheath to the mesenchymal cells in the dental follicle. Experimental studies with 1-hydroxyethylidene-1, 1-bisphosphonate (HEBP) indicate that the formation of acellular cementum must be preceded by mineralization of the mantle dentin. If the mineralization is inhibited by means of HEBP, there is precocious separation of the two layers of the epithelial root sheath after which matrix for a cellular type of cementum is formed.

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.

Orthodontically induced root resorption in rat molars after 1-hydroxyethylidene-1,1-bisphosphonate injection.

A single injection of 1-hydroxyethylidene-1, 1-bisphosphonate inhibits the formation of acellular cementum in rat molars. Instead, an atypical hyperplastic cementum is formed. In this study the resistance of this cementum to resorption was tested by applying an orthodontic force. It was found that roots lacking acellular cementum were readily resorbed. The readiness with which the atypical hyperplastic cementum was resorbed without any increase in multinucleated osteoblasts may offer useful opportunities to study the different phases of hard-tissue resorption.

Spatial and temporal repair patterns of orthodontically induced surface resorption patches.

Root resorption appears to be an inevitable sequel to orthodontic tooth movement, occurring either apically or along surfaces in and around the pressure zones. However, there is uncertainty as to the location of initial cementum repair in resorbed areas as well as the time of onset of repair. The aim of the present study was to map the spatial and temporal patterns of cementum repair in orthodontically induced areas of surface resorption following maxillary expansion. Two spatial patterns of repair of orthodontic surface resorption patches were observed with cementum deposition proceeding either from the periphery or starting somewhere in the center of the resorbing areas, although the deposition starting centrally was argued to be artifactual. Furthermore, onset of reparative cementum mineralization appeared to follow within 2 wk after release of the force, involving initially only acellular cementum formation. However, the pattern of formation changed gradually, favouring a slow deposition of cellular cementum at more advanced stages of healing.

Coronal Dentinal Nodules Induced by Single or Multiple Injections of HEBP in Young Rats

In the developing tooth, the bisphosphonate HEBP causes hypoplasias and hypomineralization of the enamel and dentine as well as inhibition of acellular cementum formation. Here, we describe a novel effect, associated with dentine mineralization. HEBP was administrated to young rats, and the maxillary molars were analyzed histologically. Localized dentinal nodules, protruding towards the pulp, were found in the developing crown of the molars. They occurred in regions, where the mantle dentine was about to mineralize at the time of the injection, and were more frequent at the mesial cusp side. The nodules accumulated mineral, as evidenced by the fluorescence after calcein and tetracyclin labelling. Histologically, the nodules were separated from the enamel by a layer of mantle dentine and were progressively surrounded by predentine and dentine. The nodules were interpreted to contain transport or metabolism intermediates, which were locally accumulated due to the interruption of the mineralization process by HEBP.

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.