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  • 1
    ISSN: 1432-0878
    Keywords: Key words Periodontal ligament ; α-Smooth muscle actin ; Osteopontin ; Bone sialoprotein ; Bone morphogenic protein ; Rat (Wistar)
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Abstract  Periodontal ligament width is precisely maintained throughout the lifetime of adult mammals but the biological mechanisms that inhibit ingrowth of bone into this soft connective tissue are unknown. As bone morphogenic proteins strongly stimulate osteogenesis and can induce ectopic bone formation in vivo, we tested the hypothesis that topical application of this powerful osteogenic agent will overwhelm the osteogenic inhibitory mechanisms of periodontal ligament cells and induce ankylosis. Wounds through the alveolar bone and periodontal ligament were created in 45 male Wistar rats. Defects were filled with either a collagen implant or collagen plus bone morphogenic protein (BMP-7), or were left unfilled (controls). Three animals per time period were killed on days 2, 5, 10, 21 and 60 after surgery for each wound type. Cellular proliferation and clonal growth in periodontal tissues were assessed by 3H-thymidine labeling 1 h before death, followed by radioautography. Cellular differentiation of soft and mineralizing connective tissue cell populations was determined by immunohistochemical staining of α-smooth muscle actin, osteopontin and bone sialoprotein. In regenerating periodontium, BMP-7 induced abundant bone formation by 21 days (2.5-fold greater than controls or collagen implant only; P〈0.001), but by day 60 the volume of the newly formed bone had returned to baseline levels and was similar for all groups. Independent of the type of treatment, periodontal ligament width was unchanged throughout the experimental period (P〉0.05). Animals treated with BMP-7 implants showed greatly increased cellular proliferation in the periodontal ligament adjacent to the wound site and in the regenerating alveolar bone at days 5 and 10 after wounding compared to the other treatment groups (P〈0.005). Animals in the BMP-7 group exhibited similar spatial and temporal staining patterns for α-smooth muscle actin, osteopontin and bone sialoprotein as controls. Collectively, these data show that BMP-7 promoted the proliferation of precursor cells in the periodontal ligament but did not induce osteogenic differentiation in this compartment. Consequently a powerful osteogenic stimulus like BMP-7 cannot significantly perturb the mechanisms that regulate periodontal ligament width and maintain periodontal homeostasis.
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  • 2
    ISSN: 1600-0765
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Medicine
    Notes: The survival rate of avulsed permanent teeth following replantation is affected primarily by the duration of the extra-alveolar period and the nature of the storage conditions. These factors are believed to strongly affect the viability of periodontal ligament (PL) cells but in vitro assays of cell viability based on vital dye assays are only weakly correlated with the tooth survival rate after replantation. The aim of the present study was to examine the relative dependence of cell membrane integrity, attachment and clonogenic capacity of human PL cells on the temperature and duration of the extra-alveolar period and the type of storage medium. Twenty-four premolar teeth were extracted for orthodontic reasons from 9 patients 11–18 years of age. Teeth were maintained at 4°C or 23°C for 15, 30, 60 or 120 min in either milk or dry conditions. Cell membrane integrity was determined by BCECF/AM dye inclusion. Plating efficiency was determined by measurement of cell attachment at 3 and 6 h. The clonogenic capacity of progenitor cells was estimated by limiting dilution and colony counts. For all assays teeth stored in milk at 4°C showed the highest percentages of BCECF positive, attached cells with clonogenic capacity. Increased storage time (15–120 min) was associated with a 50% relative reduction of BCECF staining and a 5-fold relative reduction of cell attachment regardless of storage conditions. However, the clonogenic capacity of progenitor cells decreased 25-fold over the same duration of storage. These data demonstrate that in vitro assays of clonogenic capacity are much more sensitive to extra-oral storage time and storage conditions than dye inclusion or cell attachment. We suggest that in comparison with in vitro measures of cell membrane integrity, the clonogenic capacity of PL cells is more closely linked to tooth survival rate, probably reflecting the capacity of PL progenitor cells to recolonize the root surface after replantation.
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  • 3
    ISSN: 1600-0765
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Medicine
    Notes: After severe injury to the periodontal ligament (PL), the phenotypes of cells recolonizing root surfaces influence the extent and type of repair processes. In teeth that are replanted following avulsion injury, recolonization of the PL space by osteogenic cells instead of by PL fibroblasts may favor bone formation (i.e. ankylosis) instead of PL regeneration. We consider here that recolonization processes depend in part on the storage conditions of the teeth following avulsion. We used an in vitro cell culture model to assess the effect of storage conditions on immunohistochemical staining of several marker proteins that are expressed by osteogenic cells (osteopontin and alkaline phosphatase) and fibroblasts (α-smooth muscle actin, type III and XII collagens). Prior to cell culture, extracted human premolar teeth were stored in air (“dry”) or in α-MEM (“wet”) for either 30 or 120 min as surrogate conditions for the variations of extra-alveolar tooth storage that may occur following avulsion. Collagenase/trypsin-digested suspensions of PL cells were prepared from the tissue adherent to the extracted root surface. Passage #2 or #3 cultures were immunostained and examined by fluorescence microscopy. For type XII collagen, cells from wet samples displayed perinuclear staining while cells from 30-min dry samples showed only isolated foci. The staining for 120-min dry samples was weak and non-specific. α-Smooth muscle actin was not incorporated into stress fibers in wet samples, whereas dry samples demonstrated prominent stress fibers stained for α-smooth muscle actin. Detached cytoplasmic fragments resembling cell processes that stained for α-smooth muscle actin were abundant in dry samples, indicating the presence of highly contractile cells. The staining for osteopontin was mainly perinuclear but was more intense in dry samples. The focal adhesion pattern of osteopontin staining in 120-min dry samples resembled that of migrating osteogenic cells. The pattern of staining did not vary for type III collagen or alkaline phosphatase, although staining for alkaline phosphatase was more intense in samples stored under dry conditions. We conclude that prolonged extra-alveolar dry storage favors increased in vitro growth of contractile cells expressing osteogenic cell markers while storage in cell culture medium favors growth of cells with the classical phenotype of PL fibroblasts.
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  • 4
    ISSN: 1600-0765
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Medicine
    Notes: 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.
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  • 5
    ISSN: 1600-0765
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Medicine
    Notes: Emdogain® is an enamel matrix derivative that may promote periodontal regeneration by recapitulating critical events in tooth morphogenesis. We hypothesized that Emdogain® enhances periodontal regeneration by promoting the differentiation of cells required for the synthesis of periodontal ligament, bone and cementum. Cell differentiation was examined in rat periodontal window wounds in which there is no microbial biofilm or epithelial downgrowth, thereby simplifying the model system. Defects were filled with vehicle control or Emdogain® (3 mg/ml or 30 mg/ml). Rats were sacrificed at 7, 14 and 21 d after wounding. Specimens of periodontium were immunostained for osteopontin, bone sialoprotein, osteocalcin as markers of osteogenic differentiation and for α-smooth muscle actin, a myofibroblastic marker. Morphometry and 3H-proline radioautography were used for assessment of tissue homeostasis and matrix production. Rats treated with Emdogain® (only at 30 mg/ml) showed widening of the periodontal ligament at 7 d; by 14 and 21 d, periodontal ligament width was restored to normal values for all groups. Emdogain® exerted no effect on cementum thickness, bone volume, osteoid deposition rates, or extracellular staining for osteopontin, bone sialoprotein or osteocalcin. Further, the percentage of cells with intracellular staining for osteopontin, osteocalcin or bone sialoprotein was unaffected by Emdogain®. Staining for α-smooth muscle actin was abundant in the repopulating wound but was also unaffected by Emdogain®. In conclusion, Emdogain® does not apparently affect the expression of differentiation markers or bone matrix protein synthesis in the repopulation response of wounded rat molar periodontium. Therefore the effect of Emdogain® on wound healing in the periodontium may be independent of differentiation in the cell populations examined in this model.
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  • 6
    ISSN: 1600-0765
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Medicine
    Notes: The mammalian periodontal ligament contains heterogeneous populations of connective tissue cells, the precise function of which is poorly understood. Despite close proximity to bone and the application of high amplitude physical forces, cells in the periodontal ligament (PL) are capable of expressing regulatory factors that maintain PL width during adult life. The study of PL homeostasis and PL cell differentiation requires culture and phenotypic methods for precise characterization of PL cell populations, in particular those cells with an inherently osteogenic program. Currently it is unknown if cells cultured from the PL are phenotypically similar to the parental cells that are present in the tissues. We have compared the phenotype of cells in vivo with cells derived from the PL and expanded in vitro to assess the general validity of in vitro models for the study of phenotypic regulation in vivo. Rat PL cells were isolated by either scraping the root of the extracted first mandibular molars (Group A), or by scraping the alveolar socket following extraction of first mandibular molars (Group B), or by obtaining a mixture of cells after disaggregating a block of tissue consisting of first mandibular molar, PL and the surrounding alveolar bone (Group C). Cultured cells at confluence were fixed and immunostained for α-smooth muscle actin (α-SMA), osteopontin (OPN), alkaline phosphatase (AP), or bone sialoprotein (BSP). For in vivo assessments, frontal sections of rat first mandibular molar were immunostained for α-SMA, OPN, AP and BSP. We examined osteogenic differentiation of cultured PL cell cultures by bone nodule-forming assays. In vivo and at all examined sites, 〉68% of PL cells were immunostained for AP; ∼50% and ∼51% for OPN and α-SMA (p=0.3), respectively, while only ∼8% were positively stained for BSP (p〈0.01). Analysis of cultured PL cells in Groups A, B and C showed 54%, 53% and 56% positive staining for α-SMA respectively; 51%, 56%, 54% for OPN; 66%, 70%, 69% for AP and 2.2%, 1.4% and 2.8% for BSP. The mean percentage of PL cells in situ stained for the different markers was similar to that of cultured PL cells (Group A∼Group B∼Group C in situ for p〉0.2) except for BSP which was 3 to 4 fold higher in vivo(p〈0.01). PL cell cultures treated with dexamethasone showed mineralized tissue formation for all groups (A, B, C), but no mineralized tissue formation was detected in the absence of dexamethasone. As PL cells express quantitatively similar phenotypes in vitro and in vivo, we conclude that the in vitro models used here for assessment of PL cell differentiation appear to be appropriate and are independent of the cell sampling method. Further, dexamethasone-dependent progenitors are present both on the root and bone-related sides of the PL.
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  • 7
    ISSN: 1600-051X
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Medicine
    Notes: Abstract The development of chronic inflammatory periodontal diseases is strongly correlated with the growth and maturation of subgingival bacterial colonies. Consequently a major preventive goal should be the control of plaque formation. We conducted a randomized, controlled trial to examine the short-term effect of an intensive instructional program without professional prophylaxis on the gingival health of 240. 11-14 year old school children. Plaque index (P1I), gingival index (GI), bleeding index (BI) and probing pocket depth (PD) were examined 4 × by 1 examiner blinded to the instruction. During the period of instruction, subjects in the experimental groups were involved in a plaque and gingivitis prevention program provided in separate educational sessions. One of the experimental groups (E-l: n=80) was provided with a new toothbrush, toothpaste and instruction while the second experimental group (E-2: n=80) was provided with toothbrush, toothpaste, dental floss and instruction. In the control group (C; n=80) only dental examinations were provided: no preventive program or oral health measures were conducted. Examinations were conducted every 3 months during the instructional period and at 6 months following the completion of the active preventive programme. During the experimental period there was a significant decrease (p〈0.001) in the mean P1I. GI and BI of the experimental groups following the program while in controls there was a slight but not significant increase of mean values (p 〉 0.05). During the preventive program experimental groups exhibited small but not significant (p 〉 0.05) reductions of PD. Experimental group 1 showed similar PH. GI. BI and PD scores as experimental group 2 during the study. After the instructional program was completed and a period of 6 months had passed, there was a large and significant (p 〈 0.001) increase of mean P1I. GI and BI scores in both experimental groups back to the baseline levels. We conclude that a short-term preventative program without professional instrumentation induces a transient improvement of gingival health of schoolchildren but only during the instructional period. The maintenance of improved gingival health over longer time periods requires prolonged, repeated instruction by professionals. These measures may be difficult to institute and are of questionable cost-effectivness.
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  • 8
    ISSN: 1600-051X
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Medicine
    Notes: Abstract. Several diseases as well as trauma can affect the composition and integrity of periodontal tissues leading eventually to the destruction of connective tissue matrix and cells, loss of attachment and resorption of alveolar bone, often followed by tooth loss. Replacement of the missing tooth could then be provided by endosseous dental implants healing in a form of osseo- or fibrosteal integration to the alveolar bone. Aluminium oxide ceramics, a form of endosseous implant, allows osseointegration type of healing and has demonstrated excellent biocompatibility. However, potential aluminium toxicity has been implicated in the pathogenesis of a number of clinical disorders and for this reason we examined the reproductive and mutagenic effect of aluminium trioxide ceramic implant in experimental mice. 720 female and 45 fertile male BALB-cAn NCR mice were included in the study, 3 experimental groups of fertile male mice (15 for each group) were treated with an intraperitoneal injection of aluminium trioxide (1 g/kg of body weight, group I). with ethyl-methane-sulphonate as a positive control (200 mg/kg. group II) and with Tween-80 (10 mg/kg as a negative control. Group III). Each of the labeled male mice fertilized previously uncoupled female mice during 8 weeks (a pair per week) to facilitate appropriate pre- and post-meiotic conditions of spermatogenesis to occur. Female mice were sacrificed with cervical dislocation at day 13 after fertilization. Immediately upon sacrifice the uterus was removed and the number of alive and healthy, or alive but mutated and/or dead embryos was computed to determine the dominant lethal or mutagenic effect. Animals treated with aluminium trioxide demonstrated similar effects on the reproductive and mutagenic capacity as the negative control, whereas the animals treated as positive controls exhibited significantly reduced reproductive and mutagenic capacity. Collectively, we concluded that aluminium trioxide has a very low rate of embryonal mortality and mutagenicity in mice. This finding is in general agreement with the biocompatibility of aluminium trioxide as an implant material.
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