Cyclic AMP in dental and periodontal tissues during tooth eruption in kittens.

Cyclic adenosine 3',5'-monophosphate (cAMP) concentrations and cellular distribution were studied in dental and periodontal tissues during tooth eruption in kittens. Although the mean levels of cAMP around developing teeth were similar in all the tissue samples, there were marked differences in cAMP stainability of tissues apical and occlusal to the erupting teeth.

The increase in vivo by electric currents of the 99mTc-methylene diphosphonate uptake and cyclic AMP content of the bone of the cat mandible.

Technetium-99-methylene diphosphonate (99mTc-MDP) bone images were obtained from live cats whose mandibles had been stimulated non-invasively and unilaterally with electric current (12 +/- 2 microA) for 1 week. The images were obtained using a gamma camera interfaced to a digital computer for quantitative comparison of the mandible. Radionuclide uptake by the stimulated sides increased by 59 +/- 26 per cent when compared with the contralateral control sites. Immunohistochemical staining of mandibular sections for adenosine 3',5'-monophosphate (cyclic AMP) showed that periosteal osteoblasts opposite the electrodes were intensely stained. Thus 99mTc-MDP scintigraphy is a sensitive non-invasive technique for determining in vivo enhanced bone remodelling activity. The immunohistochemistry indicates that the stimulation of bone cells was limited to surfaces adjacent to both cathode and anode.

Neurotransmitters, cytokines, and the control of alveolar bone remodeling in orthodontics.

This article described research aimed at testing the hypothesis that tissue remodeling during orthodontic tooth movement is modulated, at least in part, by factors derived from the nervous and vascular (immune) systems. Specifically, the neurotransmitters SP and VIP and the cytokines IL-1 alpha and IL-1 beta were localized immunohistochemically in paradental tissues of cat canines that had been treated by the application of an 80 g tipping force for 1 hour to 14 days. Increased staining (concentrations) of these agents were found in areas of PDL tension and compression at different time periods. Moreover, administration of SP and IL-1 beta to human PDL fibroblasts in vitro for 1 to 60 minutes resulted in significant increases in the levels of the intracellular "second messenger" cAMP, as well as of PGE2, a plasma membrane-associated fatty acid believed to serve as a local regulator of bone cell activity. Taken together, these results tend to support the hypothesis that neurotransmitters and cytokines play a regulatory role in orthodontic force-induced alveolar bone remodeling. Consequently, determination of the cytokine synthetic activity by leukocytes of orthodontic patients may inform about their alveolar bone remodeling potential.

Rapid palatal expansion in cats: effect of age on sutural cyclic nucleotides.

Previous experiments indicate that bone cells respond to externally applied stimuli with fluctuations of cyclic nucleotide levels. The objective of this experiment was to study the response of bone at the midpalatal suture to the application of tensile forces in young and old animals, through an examination of the osteoblastic staining pattern for cAMP and cGMP. The midpalatal suture was rapidly expanded for 0, 10, and 15 days, respectively, in three young and three old cats. Fresh, frozen, unfixed, undecalcified maxillae were sectioned transversely and stained immunohistochemically for cAMP and cGMP. The staining intensity for both nucleotides was increased in the osteoblasts of the young treated animals, while in the old animals the osteoblasts were only faintly stained for cAMP and cGMP. These results demonstrate that bone cells of old animals are less responsive to tensile forces than the corresponding cells in young animals.

Prostaglandin F2 alpha is associated with alveolar bone cells: immunohistochemical evidence utilizing monoclonal antibodies.

Prostaglandin F2 alpha (PGF2 alpha) has been found in mineralized tissues and may be implicated in cellular remodeling activities. The objective of this study was to localize PGF2 alpha in cat jaw bone by immunohistochemistry. Monoclonal antibodies to PGF2 alpha were prepared by the hybridoma technique and used as the first step in the tissue section staining procedure, the rabbit anti-mouse IgG conjugated to horseradish peroxidase followed by diaminobenzidine (DAB) as the substrate. Periodontal cells and cementoblasts displayed dark DAB deposits over the cell periphery. Some alveolar bone osteoblasts and osteoclasts were stained for PGF2 alpha over the cytoplasm as well as the membrane region. In the maxillary-premaxillary suture, osteoblasts on the border of the maxilla were stained very intensely for PGF2 alpha, while those adjacent to the premaxilla were stained exceptionally lightly. These results demonstrate the ability to localize PGF2 alpha in bone cells in situ and to identify intracellular sites of prostaglandin localization. Our observation suggests that PGF2 alpha may play a role in physiologic activities of fibroblasts, osteoblasts, and osteoclasts.

Guanosine 3',5'-monophosphate in bone: microscopic visualization by an immuno-histochemical technique.

Guanosine 3',5'-monophosphate (cyclic GMP, cGMP) was localized in bone cells by the use of an immunoglobulin-enzyme bridge method. We observed that in cat alveolar bone most osteoblasts did not stain for cGMP, while adjacent periodontal cells displayed cytoplasmic as well as nuclear staining. Numerous osteocytes contained diffuse reaction products over most or all of the cellular area. The method used in this study may be helpful in identifying specific hard tissue cell types whose function(s) involve cGMP.

Cellular localization and concentration of bone cyclic nucleotides in response to acute PTE administration.

The cyclic AMP and cyclic GMP concentrations of alveolar bone of control and PTE-treated cats were measured by chemical and immunohistochemical methods. In the PTE-treated animals, alveolar bone osteoblasts stained intensely for cAMP, but very weakly for cGMP; the periodontal ligament (PDL) cells stained for cAMP similarly to the controls, but some PDL cells stained more intensely for cGMP than their controls; osteocytes stained for cAMP with greater intensity than in the controls; osteoclasts stained intensely for both cyclic nucleotides. We found that bone samples taken from animals 20 and 60 min after administration of PTE contained twice the amount of cAMP, and almost three times the amount of cGMP observed in the controls. These results indicate that the cellular source of bone cyclic nucleotides in PTE-treated animals varies as to cell type, and therefore in bone and PDL the functions mediated by cAMP are not necessarily antagonistic to those mediated by cGMP.

Immuno-histochemical localization of cyclic nucleotides in the periodontium: mechanically-stressed cells in vivo.

Cyclic AMP and cyclic GMP, considered to be cell proliferation regulators, have been reported to fluctuate in proliferating fibroblasts in vitro. The objectives of this experiment were to study the localization, distribution and staining patterns of these cyclic nucleotides in mechanically-stressed, proliferating periodontal fibroblasts in vivo. Cat canines were tipped by force applications for 0 to 48 hours and serial sagittal sections of fresh frozen, unfixed, undecalcified jaws were processed immuno-histochemically for the localization of cAMP and cGMP. Periodontal tension sites were studied microscopically. Fibroblastic staining for cAMP, which was localized mainly in the cell periphery, did not change appreciably as a result of tension. However, staining intensity increased one hour after the application of force, decreased after 6 hours and increased again at 24 hours. Staining for cGMP, initially covering the entire cell area, was concentrated over nuclei three hours after onset of tension, and diffused over cell periphery and cytoplasm thereafter. Intensity of staining for cGMP was maximal at 3 hours and low at 12 and 48 hours. These results demonstrate that stress-induced fibroblastic responses in vivo involve alterations in staining intensity for both cyclic nucleotides which may correspond with fluctuations of these regulators, reported to occur in vitro in various stages of the mitotic cycle. Moreover, this technique enables the identification of the involved cells in a stimulated, non-synchronized cell population.

Immuno-histochemical localization of cyclic nucleotides in mineralized tissues: mechanically-stressed osteoblasts in vivo.

Previous experiments indicate that bone cells respond to external stimuli with fluctuations of cyclic nucleotide levels. The objective of this experiment was to study the response of alveolar bone to the application of tensile forces through an examination of the osteoblastic staining pattern for cAMP and cGMP. Cat canines were tipped by 80-g force for 0 to 48 hours. Fresh frozen, unfixed, undecalcified jaws were sectioned sagittally and stained immuno-histochemically for cAMP and cGMP. In tension sites, osteoblastic staining intensity for cAMP decreased gradually from one to three hours, and then increased by 24 hours. Intense staining for cGMP, visible in osteoblasts of all treated cats, peaked after three hours of treatment and then again at 24 hours. Generally, groups of cGMP-stained osteoblasts were found adjacent to unstained osteoblasts. The observed fluctuations in the osteoblasts staining pattern for cAMP and cGMP indicates involvement of these substances in the early response of osteoblasts to mechanical stimuli in vivo.

Substance P immunoreactivity in periodontal tissues during orthodontic tooth movement.

Neurotransmitters, such as substance P (SP), may mediate the biological response to mechanical stress applied to teeth during orthodontic treatment. This hypothesis was investigated immunohistochemically on maxillae of cats which had one maxillary canine tipped distally for a period of time ranging from 1 h to 14 days. Horizontal histological sections 5 microns thick, collected on adhesive tape while still frozen, then freeze-dried, were stained by an immunoglobulin-enzyme bridge method using rabbit anti-SP polyclonal antibodies. The specificity of the staining was evaluated either by using the serum of non-immunized rabbits in lieu of anti-SP serum, or by pre-incubating the anti-SP serum with the antigen. Only sparse SP-like immunoreactivity was detected in the dental pulp, near or in the walls of blood vessels, or in the periodontal ligament (PDL) of unstressed canines. However, the density of neuronal elements exhibiting positive staining for SP appeared to increase markedly after application of an orthodontic force. This phenomenon occurred rapidly (3 h) in the dental pulp, but later in the PDL (24 h to 14 days), mainly at compression sites. These results demonstrate that the stimulation of periodontal nerve terminals by means of orthodontic forces may induce the peripheral release of the neurotransmitter SP, suggesting that it may be an initial trigger for a biochemical cascade which comprises the activation of various types of PDL cells.

Immunohistochemical localization of cyclic AMP in the developing rodent secondary palate.

During development of the mammalian secondary palate, medial-edge epithelia (MEE) from apposing palatal shelves adhere and undergo autolysis allowing palatal mesenchymal regions to unite. In a prior study (Greene & Pratt, 1979), we reported a transient increase in levels of cyclic AMP (cAMP) in the mouse and rat palate during epithelial adhesion and cell death. The objective of this study was to examine the distribution of cyclic AMP in the developing rodent secondary palate using immunohistochemistry to localize cyclic AMP. Staining for cAMP was observed in the epithelium just prior to and during epithelial fusion (day 16 in the rat; day 14 in the mouse). Cyclic AMP was distributed throughout the epithelial cytoplasm whereas no staining was seen in nuclei. Epithelial staining for cAMP was faint or absent 24 and 48 h prior to epithelial contact. Mesenchymal staining for cAMP was minimal and associated with the plasma membrane at all stages studied. These results demonstrate that elevated levels of cAMP in the rat and mouse palate during epithelial adhesion and cell death are mainly due to increases of the nucleotide in palatal epithelium. This observation suggests that a transient increase in epithelial cAMP may play a role in palatal epithelial differentiation.

Immunohistochemical localization of epidermal growth factor in cat paradental tissues during tooth movement.

Epidermal growth factor enhances proliferation and differentiation of cells during growth, maturation, and tissue healing. The objectives were to localize the epidermal growth factor in paradental cells and to determine the effect of orthodontic treatment on its concentrations in periodontal ligament fibroblasts, alveolar bone surface lining cells, and epithelial rests of Malassez. Sixty male cats, 1 year old, were divided into 2 groups: active and sham, and further divided into 10 time groups. In the active group, 1 maxillary canine was retracted by 80 g force; in the sham group, the animals received an inactive appliance. Sagittal sections of each half maxilla were stained for epidermal growth factor; staining intensity was measured microphotometrically in 10 periodontal ligament fibroblasts, alveolar bone surface lining cells, and epithelial rests of Malassez cells in sites of periodontal ligament tension and compression, and in corresponding sites near control and sham canines. The overall mean staining intensity of the cells of the active group animals was 30.47%, whereas that of the sham group was 21.78% (P <.0001). In all 3 types, cells near the actively treated canines stained significantly darker (P <.0001) than cells near the sham or control canines, particularly between 12 hours and 7 days. These results demonstrate that orthodontic forces increase epidermal growth factor concentrations in paradental cells, suggesting that epidermal growth factor participates in the tissue remodeling that facilitates tooth movement.

Biochemical mediators of the effects of mechanical forces and electric currents on mineralized tissues.

Cyclic nucleotides (cAMP and cGMP) and prostaglandin E (PGE) have been implicated as possible mediators of the effects of external stimuli on bone cells. The objective of this study was to determine changes in relative levels of these substances in mineralized tissue cells in response to mechanical and electrical stimuli, by the use of a combined immunohistochemical-microphotometric procedure. Canine teeth of eight 10-12 month-old female cats were tipped distally with 80 g force for either 1 h or 14 days. After 1 h, a slight elevation of staining intensity in alveolar bone osteoblasts and periodontal ligament (PDL) cells was observed at sites of tension and compression. After 14 days of treatment, this effect was markedly increased. Fifteen female cats, 10-12 months old, received electric stimulation (20 micron amperes d.c.) to the gingiva of 1 maxillary canine for 1, 5, 15, 30, or 60 min. At the cathode, significant increases of staining intensity in periosteal osteoblasts for cAMP, cGMP, and PGE were found at 15 and 60 min. At the anode, a significant rise in the staining intensity of these cells for PGE was seen at 15 min; at 60 min, cGMP and PGE, but not cAMP, were elevated. These results demonstrate the usefulness of the immunohistochemical technique in detecting relative changes in mineralized tissue cell content of cyclic nucleotides and prostaglandins in response to local application of physical stimuli of short and long duration.

Electric currents, bone remodeling, and orthodontic tooth movement. I. The effect of electric currents on periodontal cyclic nucleotides.

Osteogenesis has been found to occur in response to the application of electric currents to bone. The objective of this experiment was to study the effects of D.C. electric currents on periodontal tissues in cats. Cyclic nucleotides, compounds known to be involved in cellular activation, were studied by immunohistochemistry in the involved tissues. Three groups of three young adult cats each were treated for 1, 3, and 7 days, respectively, by a device delivering 15 microamperes of direct current to bone osteoblasts and PDL cells stained intensely for cAMP and cGMP were observed adjacent to the cathode and anode, and bone apposition was found near the cathode. These results suggest that electric stimulation enhances cellular enzymatic phosphorylation activities in periodontal tissues and may be a potent tool in accelerating alveolar bone turnover.

Electric currents, bone remodeling, and orthodontic tooth movement. II. Increase in rate of tooth movement and periodontal cyclic nucleotide levels by combined force and electric current.

Piezoelectric currents in mechanically stressed bone were implicated in the activation of bone cells. The objectives of this experiment were to determine the usefulness of exogenous electric currents in accelerating orthodontic tooth movement and to study the effect of electric-orthodontic treatment on periodontal cyclic nucleotides. Maxillary canines were tipped in five cats by 80 g force. Two groups of five cats each were treated by an electric-orthodontic procedure to one maxillary canine for 7 and 14 days, respectively. Teeth treated by force and electricity moved significantly faster than those treated by force alone. Enhanced bone resorption was observed near the anode (PDL compression site), while bone formation was pronounced near the cathode (PDL tension site). Staining for cyclic nucleotides was increased when electric stimulation was added to the mechanical force. These results suggest that orthodontic tooth movement may be accelerated by the use of locally applied electric currents.

Differentiation of the avian secondary palate.

The avian secondary palate exhibits the unique feature of a midline cleft. Cryostat sections indicated that although extensive contact between homologous shelves was present, chick palatal medial edge epithelium (MEE) failed to fuse. The failure of fusion and subsequent clefting of the avian palate were correlated with continued proliferation of the avian MEE, a failure of selective MEE cell death, and an absence of elevated levels of intracellular cAMP. Moreover, immunohistochemical staining for cAMP and microspectrophotometric quantitation of staining intensity indicated that staining of chick MEE was significantly (p less than .01) less than murine MEE at comparable gestational ages. These data indicate that differentiation of the avian secondary palate is fundamentally different than reported for the mammalian palate in that many developmental events known to be associated with normal mammalian palate formation (cessation of MEE proliferation, MEE cell death, elevated levels of MEE cAMP) fail to occur in the chick. The developing avian secondary palate, with its midline cleft, thus provides an interesting and useful model system with which to compare mammalian palate formation where the palate is normally fused in the midline.