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The effect of limited drying or removal of the periodontal ligament. Periodontal healing after replantation of mature permanent incisors in monkeys.

The effect of limited drying or removal of the periodontal ligament upon periodontal healing after replantation of incisors was studied in green Vervet monkeys. All teeth were examined histologically either 2-, 4- or 8 weeks after replantation. The drying experiment demonstrated that an area of ankylosis was established after 2 weeks corresponding to the dried portion of the root and was removed by a resorptive process in the majority of cases after 8 weeks. The removal of the periodontal ligament led to development of surface-, inflammatory- and replacement resorption. Furthermore, 1 mm2 and 4 mm2 lesions on the root surface developed transient ankylosis which disappeared after 8 weeks while lesions of 9 mm2 or 16 mm2 in size resulted in an ankylosis which, in most cases, persisted after 8 weeks. Based on these findings, it is postulated that physical removal or extended drying of the periodontal ligament initiates a rapid osteogenesis in the alveolus which establishes an ankylosis. If the damaged area is placed next to a zone on the root surface with a vital periodontal ligament, a later resorptive process is established from this zone, whereby the ankylosis area is gradually resorbed. It is estimated that the extent of this process in this experiment amounted to possibly 1 -- 1.5 mm.

Animals↗

Recombinant human bone morphogenetic protein-2 stimulates osteoblastic differentiation in cells isolated from human periodontal ligament.

Periodontal ligament cells may play an important role in the successful regeneration of the periodontium. We investigated the effects of recombinant human bone morphogenetic protein-2 (rhBMP-2), one of the most potent growth factors that stimulates osteoblast differentiation and bone formation, on cell growth and osteoblastic differentiation in human periodontal ligament cells (HPLC) isolated from four adult patients. rhBMP-2 induced no significant changes in cell growth in any of the HPLCs. rhBMP-2 at concentrations over 50 ng/mL significantly stimulated alkaline phosphatase (ALPase) activity and parathyroid hormone (PTH)-dependent 3', 5'-cyclic adenosine monophosphate accumulation, which are early markers of osteoblast differentiation, in the HPLCs. rhBMP-2 (500 ng/mL) also slightly enhanced the level of PTH/PTH-related peptide receptor mRNA expression in these cells. While interleukin-1 beta enhanced ALPase activity stimulated with rhBMP-2, tumor necrosis factor-alpha inhibited the rhBMP-2-stimulated activity. Interleukin-6 induced no significant changes in ALPase activity stimulated with rhBMP-2. Although HPLCs, whether treated with rhBMP-2 or not, could not produce measurable amounts of osteocalcin, which is a marker of more mature osteoblasts, 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] induced osteocalcin mRNA expression and protein synthesis in these cells. rhBMP-2 inhibited 1,25(OH)2D3-induced osteocalcin synthesis in HPLCs at both the mRNA and protein levels. These results suggest that rhBMP-2 provides an anabolic effect on periodontal regeneration by stimulation of osteoblastic differentiation in human periodontal ligament cells, and that this stimulatory effect is differentially modulated by inflammatory cytokines during the course of periodontal regeneration.

Adolescent↗

Collagen resorption by fibroblasts. A theory of fibroblastic maintenance of the periodontal ligament.

Periodontal ligament fibroblasts contain numerous intracellular or cytosegragated collagen fibrils. These fibrils appear to be broken down within the lysosomal apparatus of the cells. There is an increase in this activity in fibroblasts associated with osteoclastic bone resorption. Cytoplasmic microfilaments and attachments to substrata indicate that the fibroblasts of the ligament have the potential of migration and cytoplasmic contraction. A theory of collagen fiber maintenance within the periodontal ligament is proposed which takes into account: (1) the motility and contractility of fibroblasts, (2) the phagocytic and degradative potential of fibroblasts, (3) the presence of intracellular collagen and (4) the reported high turnover rate of acid-insoluble collagen within the periodontium.

Actinomyces↗

The presence of ABCG2-dependent side population cells in human periodontal ligaments.

Periodontal ligament (PDL) is capable of regenerating several types of periodontal tissues. However, the stem cells in PDL have been less well characterized. Recently, it has been represented that stem cells have a potent ability to exclude Hoechst dye, and cells possessing this phenotype are termed "side population (SP)." In addition, it is suggested that the SP cells are ubiquitously present in adult tissues, therefore, we investigated the presence of SP cells in PDL. Hoechst dye efflux assay showed that PDL cells include 3.9% SP cells. This SP profile disappeared in the presence of verapamil or reserpine. Only ABCG2-expressing cells were detected in PDL cells by flow cytometric analysis. Furthermore, RT-PCR demonstrated that ABCG2 mRNA expression remarkably correlated with PDL SP phenotype. Our findings demonstrate that PDL contains ABCG2-dependent SP cells. Hence, it is possible that this population could contribute to periodontal tissue regeneration as PDL stem cells.

ATP Binding Cassette Transporter, Subfamily G, Mem↗

An osteonectinlike protein in porcine periodontal ligament and its synthesis by periodontal ligament fibroblasts.

Periodontal ligament, a soft connective tissue that lies between cementum and alveolar bone in the periodontium, has been shown to contain an osteonectinlike protein. The similarity between porcine ligament osteonectin and bovine bone osteonectin was evident from immunochemical studies, from migration characteristics on sodium dodecyl sulfate - polyacrylamide gel electrophoresis (SDS-PAGE) and from binding studies on hydroxyapatite. Using immunotransfer and immunodot analyses, ligament osteonectin was found to be extractable from tissues with 4 M guanidine-HCl (GuHCl) and 4 M GuHCl - 0.5 M EDTA and to comigrate with authentic bovine osteonectin on SDS-PAGE with a relative mass approximately 38 000. Furthermore, osteonectin from guanidine extracts of ligament was bound to hydroxyapatite in the presence of 4 M GuHCl. Immunofluorescence studies showed the osteonectin to be distributed throughout the extracellular matrix of the ligament and to be present within the ligament fibroblasts in a perinuclear, punctate distribution. Biosynthesis of osteonectin by ligament fibroblasts was studied following pulse-chase labelling with [35S]methionine and immunoprecipitation. The labelled osteonectin in the chased culture medium represented approximately 0.5% of the total labelled proteins secreted. It comigrated on SDS-PAGE with the corresponding labelled protein from pulsed cells and with the protein extracted from the tissue.

Animals↗

Characteristics of periodontal ligament subpopulations obtained by sequential enzymatic digestion of rat molar periodontal ligament.

Periodontal ligament (PDL) consists of different cell populations in various differentiation stages. In the present study, we isolated cell populations from rat molar PDL by sequential enzymatic digestion and characterized growth potential and mineralization activity of the PDL subpopulations (PDL-SP) to throw light on the mechanism of PDL remodeling and, in its turn, periodontal tissue regeneration. PDL attached to extracted rat molars was digested 2 mg/ml collagenase and 0.25% trypsin at 37 degrees C for 30 min. Then four consecutive digestions were performed for 20 min each in a fresh digestive solution. The solutions were centrifuged to collect released cells and 5 PDL subpopulations (30M-, 50M-, 70M-, 90M-and 110M-PDL-SP) were obtained. Light microscopic observation showed that about a half of PDL in width attached on the root surface of extracted teeth and 30M-PDL-SP was considered to contain cells mainly from middle portion of PDL. Scanning electron microscopic examination indicated that 110M-PDL-SP was enriched by root lining cementoblastic cells. 30M-PDL-SP showed a high level of proliferative activity. Although the growth potential of a subpopulation decreased in PDL-SP toward the root surface, 110M-PDL-SP had a high proliferative activity equivalent to that of 30M-PDL-SP. Analyses of alkaline phosphatase (ALP) and mineralization activities showed that higher activities in PDL-SP toward the surface of roots and that 110M-PDL-SP had the highest activity of ALP and the largest number of mineralization nodules. The present study shows as supposed by previous studies on cell kinetics in PDL that subpopulations with larger growth potential were generally located in the middle portion of PDL and those with higher mineralization activities toward the surface of the roots. It is suggested, however, that a possible pathway of PDL cell turnover may exist within the PDL-SP on the root surface in addition to the generally recognized pathway from the middle area of PDL to root surface.

Alkaline Phosphatase↗

Human periodontal ligament cell sheets can regenerate periodontal ligament tissue in an athymic rat model.

Conventional periodontal regeneration methods remain insufficient to attain complete and reliable clinical regeneration of periodontal tissues. We have developed a new method of cell transplantation using cell sheet engineering and have applied it to this problem. The purpose of this study was to investigate the characteristics of human periodontal ligament (HPDL) cell sheets retrieved from culture on unique temperature-responsive culture dishes, and to examine whether these cell sheets can regenerate periodontal tissues. The HPDL cell sheets were examined histologically and biochemically, and also were transplanted into a mesial dehiscence model in athymic rats. HPDL cells were harvested from culture dishes as a contiguous cell sheet with abundant extracellular matrix and retained intact integrins that are susceptible to trypsin-EDTA treatment. In the animal study, periodontal ligament-like tissues that include an acellular cementum-like layer and fibrils anchoring into this layer were identified in all the athymic rats transplanted with HPDL cell sheets. This fibril anchoring highly resembles native periodontal ligament fibers; such regeneration was not observed in nontransplanted controls. These results suggest that this technique, based on the concept of cell sheet engineering, can be useful for periodontal tissue regeneration.

Acrylic Resins↗

Collagen implants do not preserve periodontal ligament homeostasis in periodontal wounds.

An improved understanding of the differentiation of periodontal ligament cells could facilitate the development of new treatment approaches for overcoming the loss of specialized cell types caused by periodontitis. To study healing of wounded periodontal tissues and the differentiation of mineralizing connective tissue cells in periodontal ligament, we have examined the influence of wound size and collagen implantation on the regeneration of periodontium and on immunohistochemical staining for osteopontin and bone sialoprotein. Four groups of Wistar rats were wounded by drilling through the alveolar bone and by extirpation of the periodontal ligament. Wounds were 0.6 or 1.8 mm in diameter and defects were either implanted with collagen gels or were treated without implants. Rats were killed at 1 wk or 2 months after wounding and tissue sections were stained with monoclonal antibodies against rat osteopontin and bone sialoprotein. Collagen implants strongly increased staining for osteopontin and bone sialoprotein in defects at 1 wk. By 2 months alveolar bone healed completely regardless of the wound size but in large defects, periodontal ligament width was significantly reduced with or without implants. In large wounds at 2 months, collagen implants inhibited bone regeneration and there was stronger staining for osteopontin and bone sialoprotein in the bone replacing the implant, indicating that collagen prolonged bone remodelling. We conclude that implantation of exogenous collagen affects alveolar bone healing but does not preserve the width of the regenerated periodontal ligament. Therefore collagen does not appear to contribute to homeostasis in the periodontium following wounding.

Alveolar Process↗

Periodontal ligament formation around titanium implants using cultured periodontal ligament cells: a pilot study.

The aim of this study was to investigate whether a new periodontal ligament attachment will form on titanium implants when they are implanted with cultured periodontal ligament cells. Periodontal ligament cells obtained from the teeth of 3 dogs were cultured and attached to the surface of titanium implants. The implants with the cultured autologous periodontal ligament cells were placed in the mandibles of the dogs. After 3 months of healing, histologic examination revealed that, on some implant surfaces, a layer of cementum-like tissue with inserting collagen fibers had been achieved. These results demonstrated that cultured periodontal ligament cells can form tissue resembling a true periodontal ligament around implants.

Animals↗

Application of periodontal ligament cell sheet for periodontal regeneration: a pilot study in beagle dogs.

OBJECTIVE: The ultimate goal of periodontal treatment is to regenerate the damaged periodontal support. Although periodontal ligament (PDL) cells are essential for periodontal regeneration, few studies have reported the transplantation of periodontal ligament cells to periodontal defects. We developed a new method to apply periodontal ligament cells as a sheet to the defect. The aim of this study was to investigate the periodontal healing after application of the periodontal ligament cell sheet in beagle dogs. METHODS: Autologous periodontal ligament cells were obtained from extracted premolars of each beagle dog. Periodontal ligament cell sheets were fabricated using a temperature-responsive cell culture dish. Dehiscence defects were surgically created on the buccal surface of the mesial roots of bilateral mandibular first molars of each dog. In the experimental group (five defects), periodontal ligament cell sheet with reinforced hyaluronic acid carrier was applied to the defect. Only the hyaluronic acid carrier was applied to the contralateral side as a control (five defects). Eight weeks after surgery, the animals were sacrificed and decalcified specimens were prepared. Healing of the periodontal defects was evaluated histologically and histometrically. RESULTS: No clinical signs of inflammation or recession of gingiva were observed in both experimental and control groups. In the experimental group, periodontal tissue healing with bone, periodontal ligament and cementum formation was observed in three out of five defects. In the control group, such periodontal tissue formation was not observed except in one defect. Histometric analysis revealed that the formation of new cementum in the experimental group was significantly higher than that in the control group. CONCLUSION: The periodontal ligament cell sheet has a potential to regenerate periodontal tissue and may become a novel regenerative therapy.

Adjuvants, Immunologic↗

The attachment of V79 and human periodontal ligament fibroblasts on periodontally involved root surfaces following treatment with EDTA, citric acid, or tetracycline HCL: an SEM in vitro study.

OBJECTIVE: The present in vitro study has been designed to establish and compare the effects of citric acid, EDTA, and tetracycline HCl on human periodontally diseased roots on the structure, attachment, and orientation of V79 (primary Chinese hamster lung fibroblasts) cells and human periodontal ligament fibroblasts (HPDL). MATERIALS AND METHODS: Commercially available V79 cells and HPDL derived from healthy human third molars were used in this study. These fibroblasts were left in solution for seven days in order to attain confluence. Forty single-rooted teeth were obtained from patients diagnosed with periodontitis. The crown part was removed under constant irrigation and the root was split vertically into two equal halves, thus, yielding 80 specimens. Following scaling and root planing, the specimens were washed with phosphate buffered saline (PBS) and kept in 50 microg/ml gentamycin sulphate solution for 24 hours. The root pieces were then treated as follows: citric acid at pH 1, 24% EDTA, or with a 10% solution of tetracycline HCl and were then placed in V79 fibroblast cultures and HPDL cultures. The specimens were harvested after four weeks and were fixed in 2.5% glutaraldehyde in PBS before preparation for scanning electron microscopy (SEM). RESULTS: The behavior of V79 cells was similar to that of human periodontal ligament cells on root conditioned surfaces. V79 and HPDL showed a healthy morphology on root surfaces treated with citric acid and EDTA and a relatively unhealthy appearance on root surfaces treated with tetracycline HCl and distilled water (control group). CONCLUSION: The results suggest the use of citric acid and EDTA as root conditioning agents favorably affects the migration, attachment, and morphology of fibroblasts on human root surfaces, which may play a significant role in periodontal healing and regeneration.

Animals↗

Investigation of periodontal ligament reaction upon excessive occlusal load--osteopontin induction among periodontal ligament cells.

OBJECTIVE: The purpose of the present study was to investigate the reaction of the periodontal ligament to excessive occlusal loading by observing the histological changes and osteopontin induction. The possibility of ligand for receptor activator of nuclear factor kappaB (RANKL) participation in osteopontin induction was also discussed. BACKGROUND: The precise mechanism of periodontal ligament breakdown by excessive occlusal loading remains unclear. We established an experimental model for excessive occlusal loading in vivo. Osteopontin is known to be produced upon mechanical loading and is considered to induce the migration of osteoclasts to the resorption site. RANKL is one of the essential factors for osteoclast maturation and induces the constitutive induction of intracellular osteopontin in vitro. METHODS: The occlusal surface of the upper left first molars of rats was raised by steel wire bonding in order to induce occlusal trauma. The destruction of the periodontal ligament was observed and the production of osteopontin and RANKL by periodontal ligament cells was detected via immunohistochemistry. RESULTS: Our model produced wide-ranging destruction of the periodontal ligament. From day 3 to day 7, prominent compression of the periodontal ligament and osteoclast migration were observed at the apical interradicular septum. Osteopontin was detected in some osteoclasts, surrounding fibroblasts, and osteoblasts adjacent to the compression area. RANKL was observed from day 1 to day 7 around the osteoblasts and osteoclasts. CONCLUSIONS: Our model was useful for the detailed investigation of periodontal ligament breakdown during excessive occlusal loading. Although intracellular osteopontin was produced in osteoclasts with intermittent occlusal loading, the role of this protein in the cells was not clear. No correlation between RANKL distribution and osteopontin production in osteoclasts could be found.

Animals↗

[Experiments to determine the time dependent material properties of the periodontal ligament].

The periodontal ligament is a tissue that attaches the tooth (root) to its alveolar socket, and thus plays an important role in the regulation of tooth movements. Detailed knowledge of the material properties of the periodontal ligament is therefore essential to an understanding of tooth reaction to forces applied during orthodontic treatment. A knowledge of material parameters can also be used in simulations of long-term tooth movements with the aim of improving orthodontic treatment. To this end, this study investigated time-dependent material properties, namely the hysteresis behaviour of the periodontal ligament under constant-velocity loading, the influence of loading velocity on the hysteresis, and its failure under constant loading. Specimens obtained from pigs were used for testing purposes, and the experiments were conducted in a special test setup using a material testing device. The material behaviour of the periodontal ligament was shown to be viscoelastic, and the elastic parameters of material behaviour were also determined. Under constant-velocity loading, material behaviour showed a nonlinear course of the stress-strain curve, also known as hysteresis. When loading was repeated several times, the maximum stress of the hysteresis decreased with each cycle. Determination of the deflection of the specimen at different velocities showed maximum stress to be dependent on the loading rate. The measured stress-strain curves were approximated by bilinear behaviour, permitting the use of finite element calculations. Also investigated was the failure behaviour of the periodontal ligament, which revealed tissue rupture to be inconstant.

Animals↗

[Ultrastructural and cytochemical studies of clonal cell lines in fibroblasts derived from human periodontal ligaments--establishment of clonal cell lines in fibroblasts from periodontal ligaments].

It has recently been suggested that fibroblasts in periodontal ligaments have cytochemical characteristics similar to those of osteoblasts and different from those of other connective tissues. The authors isolated clonal cell lines of fibroblast-like cells from human periodontal ligaments in order to clarify their nature. Digestion with collagenase and hyaluronidase was used to isolate the cells from a human periodontal ligament. The cells were then plated in a 96-hole microplate. A single cell in a conditioned medium containing 20%FBS was placed in each hole. From these single cells large colonies ware subcultured. Subculturing was done every 8 days until more than 20 successive generations had been produced. The method developed by Lowry et al. was used to determine the ALPase activity of the cultured cells. From the 768 cells cultured from human periodontal ligament, 7 clonal cell lines were isolated in vitro. Cultures of these clonal cell lines resulted in typical, spindle-shaped, fibroblast-like cells, all of which were homogeneous. Very high ALPase activity was observed in 4 of the 7 cell lines. Enzyme reaction products occurred mainly along cell membranes. These stable clonal cell lines provide suitable systems for in vitro studies related to morphological and functional analysis of fibroblasts in the periodontal ligament.

Alkaline Phosphatase↗

Marker of cemento-periodontal ligament junction associated with periodontal regeneration.

OBJECTIVE: The purpose of this study was to identify factors promoting formation of the cemento-periodontal ligament junction. BACKGROUND: Regeneration of the cemento-periodontal ligament junction is an important factor in recovery of the connective tissue attachment to the cementum and it is important to identify all specific substances that promote its formation. To clarify the substances involved in cemento-periodontal ligament junction formation, we produced a monoclonal antibody (mAb) to human cemento-periodontal ligament junction (designated as the anti-TAP mAb) and examined its immunostaining properties and reactive antigen. METHODS: Hybridomas producing monoclonal antibody against human cemento-periodontal ligament junction antigens were established by fusing P3U1 mouse myeloma cells with spleen cells from BALB/c mice immunized with homogenized human cemento-periodontal ligament junction. The mAb, the anti-TAP mAb for cemento-periodontal ligament junction, was then isolated. The immunoglobulin class and light chain of the mAb were examined using an isotyping kit. Before immunostaining, antigen determination using an enzymatic method or heating was conducted. Human teeth, hard tissue-forming lesions, and animal tissues were immunostained by the anti-TAP mAb. RESULTS: The anti-TAP mAb was positive in human cemento-periodontal ligament junction and predentin but negative in all other human and animal tissues examined. In the cemento-osseous lesions, the anti-TAP mAb was positive in the peripheral area of the cementum and cementum-like hard tissues and not in the bone and bone-like tissues. The anti-TAP mAb showed IgM (kappa) and recognized phosphoprotein. CONCLUSION: The anti-TAP mAb is potentially useful for developing new agents promoting cementogenesis and periodontal regeneration.

Animals↗

A study of attached and oriented human periodontal ligament cells to periodontally diseased cementum and dentin after demineralizing with neutral and low pH etching solution.

BACKGROUND: This study aimed to evaluate quantitatively the migrated, attached, and oriented human periodontal ligament cells (HPLC) to periodontally diseased cementum and dentin after demineralization with low and neutral pH etching solution. METHODS: Human teeth, extracted due to periodontal diseases, were scaled and root planed so that cementum remained on one longitudinal half of the root and dentin was exposed on the other half. Forty root slices of 200 microm thickness, obtained from the mid-third of these roots, were divided into four groups: group 1, demineralized with a saturated solution of citric acid (pH 1.20); group 2, with 24% EDTA (pH 7.04); group 3, with tetracycline HCl 100 mg/ml (pH 2.00); and group 4, only scaled and root planed (control). Root slices were placed over the confluent HPLC in 35 mm culture dishes and incubated. Phase contrast microscopic photographs of attached and oriented refractile HPLC to root surfaces were taken at days 1, 3, and 7. Quantitative analysis of attached and oriented HPLC from these photographs was done using special software. RESULTS: The results showed no significant difference in the attachment and orientation index of HPLC to cementum compared to dentin in any method of demineralization at each time point (P >0.05), although there was a significantly higher cell attachment and orientation index to demineralized dentin with EDTA and citric acid than to non-etched dentin and to all 3 demineralized cementum surfaces compared to non-etched cementum (P<0.05). CONCLUSIONS: EDTA and citric acid demineralization may enhance HPLC attachment and orientation to the root surface, and it is not always necessary to remove excessive cementum when the demineralizing procedure is used.

Cell Adhesion↗