Nature and attachment of cementum formed under guided conditions in human teeth. An electron microscopic study

In an attempt at characterizing the nature and attachment of cementum formed under conditions of guided tissue regeneration (GTR) in humans, front teeth from 4 patients aged 42 to 72 years were examined at the electron microscopic level. All teeth were affected by complex periodontitis associated with advanced loss of periodontal support. Roots were surgically planed and notched, but not chemically conditioned. Either the mesial or distal surface of each tooth represented the experimental site and was covered with a biodegradable polyglactin 910 barrier, while the opposite approximal surface served as control. Following 3 months of healing, teeth were removed together with surrounding periodontal tissues including some alveolar bone. These blocks were fixed histologically, decalcified, embedded in epoxy, and sectioned for examination in the scanning (backscatter mode) and transmission electron microscope. Both experimental and control sites disclosed 2 types of regenerative cementum that seemed to be formed by cells resembling cementoblasts. The first type was characterized by a thin fringe of collagen fibrils which were arranged perpendicular to the root surface and appeared mineralized in a zone extending about 1 to 3 microm from the dentin. The second type occurred as thick patches which revealed scattered cementocytes and sheets of collagen fibrils oriented mainly parallel to the root surface, running both circularly and axially. In both situations, a continuous, thin, electrondense layer was interposed between newly formed cementum and preexisting radicular hard tissues. Interdigitation of collagen fibrils from cementum and dentin, such as observed along the natural cemento-dentinal junction, did not occur. Thus, regenerative cementum laid down in humans under guided conditions on previously diseased and planed, but not otherwise treated root surfaces shares some morphologic features with cementum formed during spontaneous repair of root resorptions. However, unlike in the course of such repair, a fibrous attachment of new cementum resembling the natural cemento-dentinal junction does not seem to be regenerated under guided conditions.     

Characterization of a collagenous cementum-derived attachment protein

We report further characterization of a cementum-derived protein that promotes the adhesion and spreading of periodontal cells. The cementum attachment protein (CAP) was extracted from bovine cementum, separated by diethylamino ethyl (DEAE)-cellulose chromatography, and purified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and C18 reverse phase high performance liquid chromatography. The purified preparation contained a single protein band migrating with M(r) 56,000. It did not cross-react with polyclonal antibodies to osteopontin, vitronectin, or other attachment proteins. The attachment activity was resistant to chondroitinase ABC digestion. An internal amino acid sequence of six peptides was determined by microsequencing, and the peptide sequences were not present in other attachment proteins described in cementum. Four sequences contained Gly-X-Y repeats typical of collagen helix. One 17 amino acid peptide had 82% homology with a type XII collagen domain. However, bovine type XII collagen did not promote fibroblast attachment. Although another 19-amino-acid-long peptide had 95% homology to bovine alpha 1 [I], two other peptides were only 74% and 68% homologous, and the CAP was not recognized by anti-type I collagen antibody. The attachment activity of CAP was susceptible to bacterial collagenase. The CAP did not cross-react with antibodies to type V, XII, and XIV collagens. These data and our previous immunostaining data indicate that the CAP is not related to other collagens or attachment proteins and that it is a collagenous attachment protein localized in cementum.     

Histologic observations on the topography of a human periodontal pocket viewed in transverse step-serial sections

A histologic overview of a periodontal pocket at the distal of a maxillary first premolar is presented. The material studied is part of the Bernhard Gottlieb collection. This material demonstrated the topography of the periodontal pocket through its complete circumference as seen in transverse step serial sections. From these observations, it is also clear that multiple pockets had formed, each involving a different area of the root surface. The pockets terminated independently and are separated from each other by areas of tissue which progress from pocket epithelium to junctional epithelium to connective tissue attachment. The existence of independent pockets around the root surface of a single tooth points to the problem of access and thus the predictability of non-surgical therapy as well as concerns that surgical access will necessarily involve areas of unaffected tissues.     

Effects of cementum-impregnated gelatine membrane on early healing following periodontal flap surgery

The purpose of this study was to investigate the initial healing response of surgically flapped periodontal tissues in the presence of gelatine membrane compounded with particles of cementum. Four monkeys with no periodontal disease were used in this experiment. Full thickness flaps were raised and recession type defects were created on the buccal side of the maxillary lateral incisors and second premolars. Exposed root surfaces were thoroughly curetted, and composite cementum-impregnated membranes placed directly onto planed root surfaces. Flaps were then sutured back to the original position. Animals were sacrificed at 2, 4, 7 and 10 post-surgical days, and block specimens including the tooth, gingiva and bone were subsequently processed for light and electron microscopy. The resultant analysis revealed that gelatine membranes were partially resorbed at 2 days post-surgery and completely resorbed by 10 postoperative days. In the early stages of gelatine resorption, most liberated cementum particles accumulated on planed dentin surface but some became demineralized within the surgical wound. Cementoblast-like cells with well-developed rough endoplasmic reticulum appeared on the root surface 7 days following surgery. Newly synthesized collagen fibrils aligned parallel to the root surface were seen at 10 post-surgical days. The results suggest that the newly developed composite membrane enhances the formation of new periodontal attachment.     

Regenerative periodontal therapy in mucogingival surgery for root coverage

This article illustrates the potential benefits of regenerative periodontal therapy in mucogingival surgery and esthetic dental treatment. Cases are described in which the treatment of soft-tissue recessions and root exposures are treated with surgical procedures where both clinical soft-tissue augmentation and the regeneration of periodontal attachment are obtained. Cases are also presented to illustrate the clinical application of guided tissue regeneration. Resorbable and nonresorbable barriers are placed over the root surface and bone and covered by the overlying flap, which allows the selective repopulation of the lesion by progenitor cells and the inhibition of a long junctional epithelium. Emphasis is placed on regenerative procedures in soft-tissue augmentation, particularly with respect to rationales, techniques, and indications.     

New ideas and advancing technology in prevention and non-surgical treatment of periodontal disease

Analytical, oral epidemiological studies in adult 'toothbrushing' populations show that the highest prevalence of missing teeth is for molars and maxillary premolars ('key-risk' teeth). The highest prevalence of subgingival microflora, gingivitis (CPITN-1), plaque retentive factors, such as subgingival calculus and restoration overhangs (CPITN-2), and diseased pockets > 3 mm (CPITN-3-4) is found on the approximal surfaces of the same teeth ('key-risk' surfaces). Primary and secondary prevention, as well as treatment of periodontal disease should be focused on these 'key-risk' surfaces. One single, well-executed subgingival scaling and root-planning procedure in deep, diseased periodontal pockets, followed by oral hygiene training and professional mechanical tooth-cleaning (PMTC) at need-related intervals, will prevent further loss of periodontal attachment. However, if the root cementum is removed during instrumentation, and the post-treatment plaque control programme fails, microorganisms will recolonize and invade the rough exposed root dentine, resulting in recurrence of periodontitis and possibly, root caries and pulpitis. Some of these problems may be overcome by the application of new instruments and methods for self-care, PMTC, removal of overhangs, scaling and root-planing without removing 'non-diseased' cementum.     

Characterization of mechanisms of interleukin-6 gene repression by estrogen receptor

Some prior reports have suggested that guided tissue regeneration (GTR) procedures achieve only partial regeneration and induces the ankylosis rather than true attachment. Accordingly, others have developed an alternative procedure employing gelatine membrane compounded with bovine cementum particles (CGM) which has proven effective in stimulating a more physiologic form of attachment. This study was undertaken to perform a direct comparison of histological results when CGM and GTR membrane were used at comparable sites in the same monkey. Three monkeys with no periodontal disease were used. Following flap surgery, recession type defects were created on the buccal side of the maxillary lateral incisors and second premolars, and the cementum was removed from the root surface at an area corresponding to the bone crest. The right and left lateral incisors and second premolars were covered with CGM and GTR membrane, respectively. The GTR membranes were removed after 4 weeks. At 6 wks, the animals were sacrificed, and specimens were prepared for histological examination. More coronally placed true new attachment was observed following application of CGM to the planed root surfaces. Application of the GTR membrane resulted in formation of bone-like cementum and ankylosis, whereas CGM established true periodontal regeneration.     

Maintenance of new cementum formed during cyclosporin A administration after suspension of the treatment

The aim of the present investigation was to examine if new cementum (NC) formed during cyclosporin A (CsA) administration was maintained after suspension of the treatment. Thirty mg/kg/d of CsA were given to 3 male Sprague-Dawley rats. Three control rats received oil-based vehicle solution. Nine wk later the drug and vehicle administration were stopped and the rats continued to be fed with the same standard laboratory diet and water ad libitum for 5 months. The rats were anaesthetized, the tissues fixed by intracardiac perfusion of fixative solution and the mandibles processed for Epon inclusion. Histological, histomorphometric and ultrastructural analysis revealed that (a) NC covered extensive areas of the root surfaces; its structural characteristics were identical to those observed in the rats killed during CsA administration. (b) collagen fibres of the adjacent connective tissue were functionally inserted into the NC. (c) In the presence of cervical NC spurs the extent of the apical downgrowth of the junctional epithelium, measured parallel to the cemento-dentinal junction, was decreased (up to 64%) compared to the one occurring in areas devoid of NC deposits. These results suggest that (a) NC deposition and its functional relations with the adjacent connective tissue are not reversible after cessation of CsA treatment and (b) in the presence of cervical NC spurs the amount of connective tissue attachment on the root surfaces is increased.     

The biologic concept for the use of enamel matrix protein: true periodontal regeneration

Guided tissue regeneration procedures have been used successfully to reestablish periodontal attachment. However, this new attachment reportedly differs from the original attachment in strength and continuity. Enamel matrix proteins secreted by Hertwig's epithelial sheath play an important role in cementogenesis on roots and in the development of the periodontal attachment apparatus. Enamel matrix protein harvested from developing porcine teeth, or enamel matrix derivative (Emdogain), is reported to induce true periodontal regeneration (the attachment of new, acellular cementum to the underlying dentin surface). The results of experimental and clinical trials of Emdogain are reviewed, and the procedure for application of the material is described.     

Cbl: complex formation and functional implications

Although the influence of tetracyclines on periodontal connective tissue cells has been the topic of many in vitro and in vivo studies, data regarding their effects on gingival epithelial cells are scarce. The present in vitro study was designed to examine the influence of minocycline, a semi-synthetic analog of tetracycline, on human gingival keratinocyte (HGK) attachment and migration. Attachment tests were performed with HGK prelabeled by tritiated amino-acids. Increasing concentrations of minocycline (10, 50, 100 micrograms/ml) in the medium produced no significant modification of cell adhesion kinetics compared to control conditions, except for 100 micrograms/ml which statistically significantly (p < 0.05) reduced the number of attached cells beyond 6 h. A 24-h cell preincubation in 10 micrograms/ml of minocycline did not alter the kinetics of HGK attachment. Scanning electron microscopic observations of attached HGK showed that the presence of 10 micrograms/ml of minocycline in the "attachment medium" induced the production of multiple filopodial extensions. Migration tests in Boyden chambers for 40 h demonstrated that HGK preincubation for 24 h in a 10 micrograms/ml minocycline-HCl solution increased significantly (p < 0.005) cell migration towards a gradient of fetal calf serum. The presence of 10 micrograms/ml of minocycline in contact with the keratinocytes in the upper compartment of the migration chambers also produced a significant (p < 0.005) result. In contrast, the presence of minocycline in the lower compartments did not produce any chemoattractive effect. Within the limits of their significance, these results suggest that, at concentrations not beyond 50 micrograms/ml, minocycline could fasten the periodontal wound coverage by epithelial cells and allow the normal reformation of a junctional epithelium.     

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.     

Effect of various demineralizing agents on mineral contents of cementum surfaces (an electron probe analysis)

The purpose of this investigation was to determine the surface morphology and mineral content of periodontally diseased cementum surfaces following root planing and conditioning with either citric acid, tetracycline HCL and minocycline HCL. Mineral contents of cementum surfaces were determined with an electron probe. All 3 demineralizing agents caused the exposure of the collagen matrix on the root surface but citric acid was the more effective. Calcium (Ca) and phosphorus (P) showed higher values in citric acid and minocycline HCL treated cementum surfaces compared to nondiseased cementum surfaces. The Ca and P were found to be in similar proportions in the tetracycline HCL treated group and nondiseased cementum surfaces. Although citric acid is a strong demineralizing agent, our specimens' cementum surfaces to which citric acid was treated showed high Ca and P content. In our opinion this difference about the effects of demineralizing agents on the surface morphology and surface element content was found to be related to the facts that whether the total amount of hypermineralized cementum as removed or not with root planing and the lacking criteria to detects the amount of this removal. Our findings suggest that these demineralizing agents can demineralize cementum surface after root planing but can not remove all of the hypermineralized diseased cementum. For this reason, it may be stated that it is more important to remove the pathologically altered cementum than applying various chemical agents.     

The epithelium-tooth interface--a basal lamina rich in laminin-5 and lacking other known laminin isoforms

The attachment of the marginal gingiva to the tooth surface is mediated by a thin nonkeratinized epithelium termed the junctional epithelium (JE). Ultrastructural studies have revealed that the attachment of the JE to the tooth surface occurs through hemidesmosomes (HD) and a basal lamina-like extracellular matrix termed the internal basal lamina (IBL). We have previously shown that neither type IV collagen nor prototypic laminin, two common components of basement membranes (BM), is present in the IBL between the epithelium and the tooth. In the present study, we show that laminin-5 is a major component of the IBL in both rodent and human tissues. By using in situ hybridization, we also show that the cells of the JE express the LAMC2 gene of laminin-5. In other parts of gingival epithelium, LAMC2 gene expression is less prominent. Our results indicate that the epithelium-tooth interface is a unique structure wherein epithelial cells are induced to secrete a basal lamina containing laminin-5 and no other presently known laminin isoform.     

Risk factors for premature birth. I. Analysis of selected factors in a group of non-working women

The distribution of glycosaminoglycans in the extracellular matrix of human cementum was investigated in periodontally involved and periodontal disease-free teeth separated into eight different age groups (from 12 to 90 years), to investigate possible changes in the distribution of glycosaminoglycan species associated with ageing and periodontal disease. A standard indirect immunoperoxidase technique was used, with a panel of monoclonal antibodies, 2B6, 3B3, 5D4, and 7D4, that recognize epitopes in chondroitin-4-sulphate/dermatan sulphate (C-4S/DS), chondroitin-6-sulphate (C-6S), keratan sulphate (KS) and a novel sulphated chondroitin sulphate (CS) epitope, respectively. Intense positive staining for C4-S/DS was observed at the margins and lumina of almost all the lacunae and canaliculi in cellular cementum in all sections. Immunoreactivity to C6-S, KS and novel CS epitopes was limited to a proportion of lacunae and canaliculi in all sections, although C6-S and the novel CS epitopes were more widely distributed than KS. In acellular cementum, there was no demonstrable staining for any of the glycosaminoglycans except where periodontal ligament (Sharpey's) fibres insert; periodontal ligament fibres inserting in cellular cementum also demonstrated positive immunoreactivity. In addition, the cementoblasts on the outer root surface, as well as the pericellular areas around a proportion of these cells, demonstrated positive immunoreactivity. These results indicate that glycosaminoglycan species present in human cementum include C4-S, DS, C6-S, and novel sulphated CS epitopes. KS is also present in cementum but is limited to a more restricted proportion of lacunae and canaliculi. Regional differences in the distribution of glycosaminoglycans exist between the two cementum types, but no qualitative differences in that distribution were observed between the various age groups or between periodontally involved and periodontal disease-free teeth. The immunoreactivity observed in a proportion of lacunae after staining for C6-S, KS, and novel sulphated CS epitopes could suggest the existence of different cementocyte subpopulations.     

Effects of resorbable membrane placement and human osteogenic protein-1 on hard tissue healing after periradicular surgery in cats

Periradicular surgeries were performed on the maxillary cuspid teeth of twelve cats. Before reapproximation of the surgical flaps, eight of the osteotomies were covered with a resorbable membrane and eight were filled with human osteogenic protein-1 (hOP-1) on a collagen carrier. The remaining eight sites received no further treatment and served as controls. The animals were euthanized after 12 wk, and the specimens were examined histomorphometrically for the presence or absence of osseous regeneration, inflammation, and cementum formation on the root ends. The results showed that the sites treated with the membrane exhibited significantly more inflammation adjacent to the resected root ends (p < 0.05), and that the use of the membrane had no statistically significant effect on osseous healing or new cementum formation. The use of hOP-1 was associated with a significant decrease in the thickness of new cementum formed on the resected root ends (p < 0.05), but had no statistically significant effect on osseous healing or degree of inflammation. Based on these results, it seems that neither the use of hOP-1 nor resorbable membranes have a positive effect on periradicular tissue healing in endodontic surgery.     

Characterization of the ntrBC genes of Azospirillum brasilense Sp7: their involvement in the regulation of nitrogenase synthesis and activity

The in vitro effect of citric acid application on periodontally involved root surfaces was investigated using scanning electron microscopy. Teeth were selected following specific criteria. The specimens were prepared for observation, and the features of the root surface were studied. The results demonstrated that a 3-minute application of citric acid (pH = 1.0) to the root surface after scaling and root planing demineralized the outer root surface, opened the dentinal tubules, and exposed collagen fibers. Scaling and root planing alone left the surface with a smear layer, scattered islands of cementum, and no collagen fibers or exposed dentinal tubules.     

Human monocyte response to cementum extracts from periodontally diseased teeth: effect of conditioning with tetracycline

Monocyte inflammatory cytokines, such as TNF alpha and IL-1 beta, have been implicated in the pathogenesis of periodontal destruction. The present study was designed to test the ability of extracts of cementum from periodontally diseased teeth to induce the secretion of these mediators by monocytes, to evaluate the role of adsorbed endotoxin in this process, and to test the effect of cementum conditioning with tetracycline on the monocyte response. Human monocytes were incubated with varying concentrations of cementum extracts, and TNF alpha and IL-1 beta levels in the media were measured. The results showed that while extracts of healthy cementum had no effect on monocyte secretion, concentration as low as 0.5 mg/ml of cementum from diseased sites raised the levels of TNF alpha and IL-1 beta secretion 10-fold. This response was dose-dependent. Diseased cementum were found to contain 1.5 ng/mg endotoxin, while endotoxin was not detectable in the extracts of the healthy cementum. However, neutralization of the endotoxin by polymyxin B only partially reduced the monocyte secretory response by 50 to 70%, suggesting that other factors in the extracts are also involved in monocyte stimulation. To simulate the effect of root conditioning, cementum was first agitated in a tetracycline or control solution prior to its extraction in media. Pretreatment of diseased cementum with tetracycline (50 mg/ml) was found to block the secretion of TNF alpha from cementum-stimulated monocytes. Pretreatment of the diseased cementum with 10 mg/ml tetracycline was not more effective than saline and HCI controls, with all treatments reducing cytokine secretion by approximately 80%. The direct addition of tetracycline to cementum-stimulated monocyte culture was found to block TNF alpha secretion in a dose dependent manner. The results suggest that extracts from diseased cementum are potent stimulators of monocyte secretion, and that endotoxin as well as other factor(s) appear to be involved. These factors are partially extracted by washing and a 10 mg/ml tetracycline solution is not more effective than saline in achieving this goal. In addition, tetracycline was found to be a potent inhibitor of TNF alpha secretion by cementum-stimulated monocytes, suggesting a novel mechanism for this drug in periodontal therapy.     

Localization of uncalcified cementum in adult rat molar roots and its relation to physiological tooth movement

The study was designed to elucidate the effect of physiological tooth movement on cellular cementum, using the upper molar roots of 10-week-old rats. Paraffin sections stained with haematoxylin and eosin displayed two types of cellular cementum, lightly and darkly staining. The lightly stained was present on the distal half of all molar roots except the mesial root of the first molar. The alveolar bone facing the lightly stained cementum showed resorption lacunae and multinucleated osteoclasts, while the opposite bone surface was lined with osteoblasts. In contact microradiographs of undemineralized ground sections, the X-ray density of the lightly stained cementum was similar to that of the periodontal ligament and pulp, while the X-ray density of the darkly stained cementum was similar to that of alveolar bone. Tetracycline labelling lines were seen at the interface between the two types of cellular cementum as well as on surfaces of bone and cementum located mesially to the root dentine. The results suggest that the mechanical stress of tooth movement differently affects the alveolar bone and cellular cementum; the bone is resorbed whereas the cementum resists resorption and its calcification is inhibited under the compressive force of tooth movement.     

Effect of non-erupted third molars on roots of approximal teeth. A radiographic, clinical and histologic study

This study was undertaken to evaluate clinically and histologically root resorption in extracted human second molars in close proximity to non-erupted third molars. The control group consisted of extracted second molars that were proximal to fully erupted third molars. Eight out of the 11 teeth in the study group presented different degrees of radiographic root resorption, nine presented clinical resorption, and all 11 had histologic evidence of root resorption. In the control group, no signs of root resorption were seen radiographically or clinically. Histologically, limited sites of resorption were identified in all teeth, which were partially repaired by cellular cementum. Histologic observation of study specimens revealed root surface resorption in 10 out of the 11 teeth, one showing replacement resorption as well. Inflammatory resorption was observed in the three most advanced cases in the study group. Reparative cementum partially lining resorbed areas was evident in all teeth with surface resorption. Within the limits of this study, radiographic identification of distal root resorption of second molars in close proximity to non-erupted third molars appears reliable. The findings may support the hypothesis that the presence of a non-erupted third molar in close proximity to the distal root of the second results in root resorption.