A collagenous cementum-derived attachment protein is a marker for progenitors of the mineralized tissue-forming cell lineage of the periodontal ligament.

The periodontal ligament (PDL) is a fibrous and cellular connective tissue that mediates tooth attachment to bone, and it comprises fibroblastic and mineralized tissue-forming (MTF) progenitors. The MTF progenitors are believed to give rise to the cementoblastic and osteoblastic lineages. Cementum attachment protein (CAP) is a collagenous cementum-derived protein which binds strongly to osteoblasts, moderately to PDL cells, and weakly to gingival fibroblasts. The aim of the present study was to determine the relationship between the capacity of PDL progenitors to bind CAP and their potential to express alkaline phosphatase (ALP) and form mineralized-like tissue in culture. Cloned human PDL progenitor populations obtained from nine human donors were assayed for their constitutive capacity to bind CAP and express ALP, and for the dexamethasone-induced potential to form mineralized-like tissue in culture in the presence of ascorbic acid and beta-glycerophosphate. Forty percent of the progenitor clones produced mineralized-like tissue. Two patterns of mineralization were observed: a spread and flat pattern similar to that produced by human bone cells in culture and a nodular ridge-like type resembling that formed by human cementoma-derived cells. A direct correlation was found between the percentage of ALP positive cells in each progenitor clone and the amount of mineralized-like tissue formed (r = 0.565). Similar correlations were found between the number of ALP positive cells and the binding capacity of each clone (r = 0.392) and between the CAP binding capacity and mineralized-like tissue formation (r = 0.584). Multiple regression analysis indicated that the constitutive capacity of a clone to bind CAP and express ALP is directly correlated to its dexamethasone-induced potential to form mineralized tissue (r = 0.675). These results indicate that CAP binding and ALP expression can serve as markers for the identification of MTF progenitors in the heterogeneous cultured population of the human periodontal ligament. These data show for the first time that binding capacity to extracellular components of mineralized tissues can be a marker for mineralized tissue-forming progenitors.

The odontoblast process extends to the dentinoenamel junction: an immunocytochemical study of rat dentine.

The length and extent of the odontoblast cell process in dentine has been the subject of controversy for many years. Here an immunofluorescence technique has been applied at the light microscope level to rat coronal dentine to localize the intracellular components actin and tubulin. Adult rats were perfused with periodate-lysine-paraformaldehyde fixative, teeth were extracted, the molar crowns were demineralized, dehydrated, wax embedded, and 6 micron sections were prepared. The sections were postfixed in -20 degrees C acetone and then incubated with affinity-purified rabbit anti-actin or anti-tubulin antibodies, followed by fluorescein-conjugated goat anti-rabbit immunoglobulin. Intratubular immunofluorescence labeling for tubulin extended to the dentinoenamel junction, whereas labeling for actin, although extending to the dentinoenamel junction, was more prominent in the pulpal third of the rat dentine. Areas in which odontoblast processes are known not to occur, i.e., the atubular dentine, were not labeled by either antibody. The presence of actin- and tubulin-containing structures extending to the dentinoenamel junction is consistent with the hypothesis that the odontoblast process traverses the dentine for up to 3-4 mm, all the way to the dentinoenamel junction. Furthermore, the different staining patterns for actin-containing microfilaments as compared to tubulin-containing microtubules suggest that these two filamentous systems may have different roles in the function of the odontoblast process.

The effects of partial demineralization and fibronectin on migration and growth of gingival epithelial cells on cementum in vitro.

The capacity of mineralized cementum to support epithelial cell migration and growth and the effect that fibronectin and partial demineralization of cementum have on these processes were assessed in vitro. Dog gingival explants, 1 X 2 mm, were cultured on the cementum surfaces of pig root pieces in a defined medium consisting of DMEM and F12 (1V/1V), transferrin, insulin, epidermal growth factor, cortisone, selenium, and high-density lipoprotein. Sixty root pieces were divided into four equal groups according to the treatment: (1) untreated mineralized cementum; (2) treated with 5 micrograms of fibronectin; (3) partially demineralized in 18% EDTA for 30 min; and (4) both partially demineralized and fibronectin-treated as above. Epithelial cell migration and growth on each of the four differently treated cementum surfaces were assessed histomorphometrically by means of scanning electron microscopy. The defined culture medium supported the selective migration and growth of epithelial cells from the gingival explants onto the mineralized cementum. This was confirmed by the positive immunostaining of these cells with antikeratin antibodies. Partial demineralization of cementum inhibited epithelial migration and growth by 83% and 91%, respectively. Fibronectin treatment did not affect epithelial cell migration and growth on mineralized cementum, but it decreased the degree of epithelial cell migration and growth inhibition on partially demineralized cementum to 57% and 43%, respectively. The results indicate that: (i) mineralized cementum may consist of components that are recognized by gingival epithelial cells and support their migration and growth in vitro; (ii) these components can be removed by demineralization; and (iii) fibronectin partially restores epithelial cell migration and growth on partial demineralized cementum in vitro.

Bacterial endotoxin inhibits migration, attachment, and orientation of human gingival fibroblasts in vitro and delays collagen gel contraction.

The purpose of this study was to assess the effect of endotoxin adsorbed to dental surfaces and to collagen type I on the migration, attachment, and orientation of human gingival fibroblasts (HGF). Transversely cut porcine tooth root slices (RS), 200 micron thick, were prepared. Half of the RS obtained were partially demineralized in EDTA. Half of the demineralized and non-demineralized RS were incubated with 400 micrograms/mL of endotoxin for 24 hr, whereas the other half were maintained in PBS and served as controls. Experimental and control RS were placed on confluent layers of HFG and cultured for six days. Cell migration toward and cell attachment to the periphery of the RS and the formation of oriented cell sheets were assessed by means of photographic techniques. Additionally, six-day-old cultures were fixed and processed for SEM observation. In separate experiments, the effect of endotoxin on cell attachment to collagen type I and on contraction of three-dimensional collagen gels was assessed. It was found that: (i) bacterial endotoxin inhibited migration and attachment of HGF to both demineralized and non-demineralized cementum and interfered with the development of oriented cellular structure: (ii) the inhibitory effect was significantly more pronounced for non-demineralized than for demineralized cementum: (iii) the morphology of HGF attached to endotoxin-treated dental surfaces was altered compared with that of their controls: and (iv) bacterial endotoxin inhibited cell attachment to collagen type I and delayed the contraction of collagen gel.

Mechanism of endotoxin inhibition of human gingival fibroblast attachment to type I collagen.

Bacterial endotoxin inhibits the attachment of human gingival fibroblasts to collagen. The present study attempted to elucidate the possible mechanism of this inhibition. Two mechanisms were considered: direct toxicity to the cells and steric interference. Collagen substrates were prepared by rat type I collagen being air-dried in the wells of 24 multi-well plates. Experimental collagen substrates were treated with 50 micrograms of endotoxin/well, while untreated collagen substrates served as controls. Two mL of cell suspension (10(4) cells/mL) was added to each well, and these were incubated at 37 degrees C for two h. The average cell number/mm2 attached to experimental and control substrates was determined. Cell attachment to endotoxin-treated collagen was inhibited by 78%, compared with that to untreated collagen. The washing of the endotoxin-treated collagen for two h did not affect the inhibition of cell attachment, whereas after 24 h of washing, cell attachment was inhibited by 54%, compared with that to untreated collagen. Pre-incubation of the cells in endotoxin for two h did not affect their attachment to collagen. The addition of fetal calf serum (15%) to the experimental system completely reversed the inhibition of fibroblast attachment to endotoxin-treated collagen. These findings suggest that endotoxin interferes with fibroblast attachment to collagen through a steric phenomenon, possibly by blocking the binding sites on the collagen molecule recognized by the membrane receptor for collagen.

The effects of titanium and hydroxyapatite on osteoblastic expression and proliferation in rat parietal bone cultures.

Titanium and hydroxyapatite are used for the fabrication of dental and orthopedic implants. The longevity of these implants depends on the amount and rate of bone formation that occurs around their surfaces. In the present study, the effects of titanium, hydroxyapatite, and polystyrene (control) on the proliferation of rat calvarial cells, and on their capacity to express alkaline phosphatase and respond to parathyroid hormone (PTH) stimulation, were studied. The nature of the substrate did not affect the DNA and protein contents of experimental and control cultures throughout the experimental period. Alkaline phosphatase expression and PTH response, as assessed by DNA synthesis and adenylate cyclase activity, were higher in cultures grown on hydroxyapatite and polystyrene than in those grown on titanium. These results indicate that hydroxyapatite was a more favorable substrate than titanium for the growth and differentiation of osteoblast-like cells in vitro.

Changes in the shape and orientation of periodontal ligament fibroblasts in the continuously erupting rat incisor following removal of the occlusal load.

One of the main theories which attempts to explain the phenomenon of tooth eruption suggests that periodontal ligament (PDL) fibroblasts move actively and pull the tooth with them out of its socket. To find further support for this theory, we determined the changes in the shape and orientation of PDL fibroblasts induced by a transition from impeded to unimpeded eruption. We measured nuclear area, elongation (length-to-width ratio), and orientation (angulation in relation to the eruption axis) of PDL fibroblasts in impeded (functionally loaded) and unimpeded (hypoloaded) rat incisors. The mean cross-sectional nuclear area did not differ between fibroblasts in the two groups. In contrast, unimpeded eruption resulted in a marked increase in the mean nuclear elongation (from about 2 to 2.56) and a significant increase in the mean nuclear orientation (from 25.6 to 14.0 degrees). Bivariate analysis suggested that these changes occurred in the same cells. Analysis of nuclear elongation and orientation at various distances from the cementum toward the alveolar bone revealed a profile of both parameters, such that cells located 20 to 80 microns away from the cemental surface were more elongated and more frequently oriented toward the eruption axis, while cells at 0 to 20 and 80 to 100 microns were more round/oval and had a greater angulation with the eruption axis. These findings, together with other observations of changes in cell number, number of microtubules, and migration velocity which occur on the shift to unimpeded eruption, support the theory of active movement of PDL fibroblasts as an important component of tooth eruption.

Role of attrition and occlusal contact in the physiology of the rat incisor: IX. Impeded and unimpeded eruption in lathyritic rats.

In the rat, the administration of a lathyrogenic agent reduced both impeded and unimpeded eruption rates of incisors. Unimpeded eruption rates were greater than impeded eruption rates. The general eruption pattern in the experimental rats was, however, similar to that in the control rats. Thus, eruption was possible even in rats with a lathyrogenically impaired periodontal ligament.

Role of attrition and occlusal contact in the physiology of the rat incisor: X. The part played by the periodontal ligament in the eruptive process.

Removal of the proliferating base of the rat incisor did not influence the rate of eruption which responded to changes in impediment to eruption in a fashion similar to that for control teeth. It is the peridontal ligament rather than the proliferating cells that is responsible for tooth eruption. The elements of the peridontal ligament apparently responsible for tooth movement are the mature fibroblasts.

Immunolocalization of a human cementoblastoma-conditioned medium-derived protein.

Little is known about the molecular mechanisms that regulate the cementogenesis process, because specific cementum markers are not yet available. To investigate whether a cementoblastoma-conditioned medium-derived protein (CP) could be useful as a cementum biological marker, we studied its expression and distribution in human periodontal tissues, human periodontal ligament, alveolar bone, and cementoblastoma-derived cells. In human periodontal tissues, immunoreactivity to anti-CP was observed throughout the cementoid phase of acellular and cellular cementum, cementoblasts, cementocytes, cells located in the endosteal spaces of human alveolar bone, and in cells in the periodontal ligament located near the blood vessels. Immunopurified CP promoted cell attachment on human periodontal ligament, alveolar bone-derived cells, and gingival fibroblasts. A monoclonal antibody against bovine cementum attachment protein (CAP) cross-reacted with CP. These findings indicate that CP identifies potential cementoblast progenitor cells, is immunologically related to CAP species, and serves as a biological marker for cementum.

Growth and migration of gingival epithelial cells on mineralized and partially demineralized root surfaces in an in vitro system.

The capacity of epithelial cells to migrate and grow from gingival explants cultured on mineralized and partially demineralized root surfaces in an in vitro system was assessed. Explants of attached gingiva were obtained from mongrel dogs, cut into rectangular pieces (1 x 2 mm) and cultured either on mineralized or partially demineralized cementum in a defined culture medium containing transferrin, insulin, epidermal growth factor, cortisone, high density lipoprotein and selenium. After seven days of culture, the specimens were prepared for scanning electron microscopic examination. The amount of epithelial outgrowth from each explant was assessed by measuring the distances between the four aspects of the rectangular explant and the furthest epithelial cell located opposite to each of these aspects. The mean value obtained for epithelial outgrowth of explants grown on mineralized cementum was six times higher than that for explants cultured on partially demineralized cementum. These results indicate that partially demineralized cementum does not support epithelial growth and migration in vitro.

Partial regeneration of periodontal tissues using collagen barriers. Initial observations in the canine.

The capacity of collagen membranes to support guided regeneration of periodontal tissues in the dog was assessed. The mesiolabial, labial and distolabial aspects of the mesial root of the second and third mandibular premolar were surgically exposed in three beagle dogs. Collagen membranes, 0.5 to 0.7 mm thick, prepared from a purified solution of rat-Type I collagen were interposed between the gingival flap and the exposed root surfaces of the right premolars. The left premolars were sham-operated without the use of collagen membranes. Animals were killed one month after surgery. Tissue blocks, including the surgical sites, were removed and prepared for histological and histometric examination. Long epithelial attachment was the modality of healing in the control sites. The apical level of the junctional epithelium was located either at, or close to, the apical level of the defect. The experimental sites exhibited a combination of three healing modalities: (1) partial regeneration of periodontal tissues (new bone, periodontal ligament and cementum) occurred in the apical half of the defect, (2) long epithelial attachment developed in the coronal quarter of the defect and (3) connective tissue adhesion developed between the two. Pocket depth was similar in both the control and experimental sites. Collagen membranes could not be identified at the time of examination. The results indicate that: (1) collagen membranes have the capacity to support regeneration of periodontal tissues and (2) collagen membranes are either incorporated within the healing tissues or degraded by these during the healing process. These findings suggest that collagen membranes may be of value in reconstructive periodontal therapy.

Heparan sulfate and fibronectin improve the capacity of collagen barriers to prevent apical migration of the junctional epithelium.

The objective of the present study was to assess the effect of bilayered/collagen barriers enriched with fibronectin and heparan sulfate on the prevention of apical migration of the epithelium during the initial stage of periodontal wound healing. Experimental osseous defects were produced on the labial aspect of maxillary canines in dogs. Experimental sites were treated with either bilayered enriched collagen barriers or with non-enriched bilayered collagen barriers, using the guided tissue regeneration technique. Control sites were treated with monolayered collagen barriers that were not enriched with fibronectin and heparan sulfate. Histologic and histomorphometric examinations performed on specimens obtained 20 days post-operative indicate the formation of a short junctional epithelium in the experimental sites treated with enriched collagen barriers. In this group, 95% of the occlusal-apical length of the defects was repopulated by connective tissue cells. In the other 2 groups, a long junctional epithelium developed with only 65% of the occlusal-apical length of the defects being repopulated by connective tissue cells. These findings suggest that the enrichment of collagen barriers with fibronectin and heparan sulfate may be important to enhance the repopulation of exposed root surfaces by connective tissue cells and prevent the apical migration of the epithelium during the initial stages of periodontal wound healing.

Observations on a new collagen barrier membrane in 16 consecutively treated patients. Clinical and histological findings.

BACKGROUND: Space-maintaining capacity, cell disclusive potential, and stability over time are crucial factors to achieving sufficient bone augmentation with membrane barriers. The case series presented here assessed a new collagen barrier used in bone augmentation. Clinically, the healing pattern, especially in cases of secondary healing, was studied. METHODS: Soft tissue healing was documented by photographs, and the size of the dehiscences calculated by image analysis. The measurements were performed on digitized photographs. During reentry, barrier remnants were dissected and histologically evaluated. RESULTS: The mean value for dehiscences was 35.5 mm2; all dehiscences healed within 4 weeks after the exposure became evident. The difference was statistically significant between the week 2 and week 6 visits (P = 0.008) for each previously exposed site. The histologic observation of barrier remnants revealed direct apposition of fibrous and bone tissues on the membrane surface. CONCLUSION: In cases of membrane exposure, gingival dehiscences always disappeared in the following weeks without affecting the healing process. Histologic results showed barrier stability over a 6-month period, promoting bone regeneration.

The effect of toxic doses of 1,25-dihydroxycholecalciferol on dental tissues in the rat.

Vitamin D-depleted rats 4-weeks old were divided into three groups and given daily for 5 weeks cholecalciferol (0.25 microgram) or 1,25(OH)2D3 (0.075 microgram). The third group received no treatment with vitamin D sterols. A fourth control group was fed a diet containing vitamin D. The animals were killed after 5 weeks, plasma was prepared for calcium analysis, and incisors and molars were taken for histology. Growth was monitored throughout. Plasma calcium, body weight and the physical condition of the 1,25(OH)2D3-treated animals indicated that they were toxemic. The pulp-dentine complex of their incisors showed premature aging of fibroblasts and odontoblasts, disturbances in the dentinal matrix and osteodentine formation. That of molars was not affected. There was hypercementosis and bone-like tissue formation in the periodontal-ligament which in the incisors was considerably enlarged; some molars were ankylosed. The pulp-dentine complex of the incisors and molars of the rats in the remaining three groups appeared normal except for zones of hypomineralization in incisors of the third group. The supporting tissues of the teeth of the rats in the other three groups were within normal limits. Thus toxic doses of 1,25(OH)2D3 affected the dental tissues of both developing and mature teeth.

Partial pulp resection increases unimpeded eruption rates of the rat incisor.

Thirty rats were divided into 3 equal groups. Following 9 days of induced unimpeded eruption, a single partial pulp resection was performed on the repeatedly-shortened lower left incisor of one group. A total of 3 resections, each repeated after 48 h, was performed on the animals in a second group. The lower left incisor was shortened but the pulp was not resected in the 3rd sham-operated control group. Eruption rates of all of the incisors were recorded daily. Partial resection of the pulp increased the unimpeded rate of the experimental incisors in the first group by 50 per cent at 24 h after operation and an increase occurred 3 times in the second group of rats (by 45, 36 and 25 per cent respectively). The rate returned to control levels 24 h later. No significant differences were observed between the eruption rates of the right maxillary and mandibular incisors of the animals in the 3 groups. The increased rate of eruption may be induced by local regulators from the traumatized pulp that are transmitted to components of the periodontal ligament which provide the force for eruption.

Formation of new periodontal attachment apparatus after experimental root isolation with collagen membranes in the dog.

This study evaluated the ability of collagen membranes to act as biodegradable barriers that interfere with colonization of the root surface by gingival cells and allow selective repopulation of the denuded root surface by periodontal ligament-derived tissue. Over a 3-year period, experimental and control surgical procedures were performed on canine teeth in six beagle dogs and on premolars in three beagle dogs. Results showed that the membranes partially prevented apical migration of epithelium during healing. Regeneration of new cementum, alveolar bone, and periodontal ligament-like tissue was found in the studied premolars but notably absent on the canines.

The development of a novel implant: induction of a non-rigid and self-renewing anchorage of artificial implants to bone.

Artificial implants currently used in orthopaedic surgery and dentistry are anchored to the surrounding bone by rigid mechanical fixation. Long-term studies indicate that the rate of implant failure due to loosening increases steeply after 10 years of function. The loosening is attributed to the micro-movements occurring at the bone implant interface. Non-rigid, self-renewing ligamentous anchorage is nature's solution to the problem of micro-movements. An excellent example of this type of anchorage is the tooth-bone system, where the tooth is anchored to the bone by a fibrous connective tissue. A novel artificial implant, bearing on its surface a unique biological substrate (BS), was designed to induce a ligamentous anchorage of implant to bone. The implant consists of a metallic core to which the BS is bound. The BS is a composite of plastic material and a collagen mesh which is partly incorporated into the plastic material and partly freely extended from its outer surface as artificial Sharpey's fibers. BS fabrication did not affect the capacity of the collagen to withstand non-specific degradation in vitro. Non-weight-bearing implants implanted into the femoral bone of rats induced and maintained a ligament-like tissue up to 4 months. The collagen fibers of the ligament-like tissue were spliced with the artificial BS Sharpey's fibers and were also anchored as Sharpey's fibers into the surrounding bone. Examination of control plastic implants (without the BS) revealed bone formation in close approximation to the implant surface.(ABSTRACT TRUNCATED AT 250 WORDS)

On the derivation of cell kinetics from histomorphology, observed in the rat incisor odontoblast.

A method to extract cell kinetic information from histomorphology is presented. Each replicating tissue is essentially an ordered structure with an origin where cells are formed and a periphery toward which they are displaced. The displacement path is called the tissue radius. The tissue variables may be studied in two domains, space and time. The first embraces all the states a cell may assume while the second specifies the cell transition rates. During steady state both domains are related linearly. These ideas are illustrated in the rat incisor odontoblast population whose life expectation is determined by the tooth wall shape. The odontoblast cell population paves the interior of the tooth wall delimiting a cone-shaped pulp. Near the root apex the dentine wall is barely visible. As one proceeds distally, the wall thickens while the pulp narrows. Pulp narrowing is associated with odontoblast cell loss whose magnitude may be deduced from the change of the pulp circumference CI(x) (x is the distance from tooth origin). The odontoblast force of mortality mu(x) may be calculated from the instantaneous perimeter change:mu(x) = - CI'(x)/CI(x); where CI'(x) stands for the derivative of CI(x). This equation serves for the construction of the odontoblast life table which may be studied in space and time.