Vascular endothelial growth factor and e-nitric oxide synthase-mediated regenerative response occurring upon autologous and heterologous bone grafts.

Bone regeneration procedures allow oral rehabilitation with dental implants also in edentulous ridges with severe bone atrophy. The integration of grafted materials with the host tissue can initiate regenerative, inflammatory and apoptotic response. Since molecular mechanisms exist at the basis of such response, the aim of this work is to investigate, by immunohistochemical analyses, the expression of proteins involved in the graft integration process, in parallel to clinical and histological modifications, occurring on sites treated with extraoral autologous bone graft deriving from the parietal region of the calvaria (eAB), intraoral autologous bone graft deriving from mandibular ramus (iAB) and heterologous bone graft from swine (hB) in human patients. In our study, the immunohistochemical expression of BSP, VEGF, eNOS in eAB samples was significantly higher (p < 0.05) compared to values recorded in iAB and hB samples. The inflammatory response, investigated by iNOS expression, was found lower in all autologous samples (eAB and iAB) compared to hB, at statistically significant values. Moreover, the expression of the pro-apoptotic molecule, Bax, resulted significantly lower (p < 0.05) in eAB than in iAB and hB samples. These values, together with the low number of apoptotic cells detected in autologous samples, suggest a good regenerative response when extraoral autologous bone graft is used in comparison to the response from the other grafts, and also suggest the use of calvaria graft as a predictable therapeutic procedure for repairing severe bone defects in oral and maxillofacial surgery, not only by clinical and biomechanical criteria, but also from a biomolecular aspect.

Expression of nitric oxide synthases in orthodontic tooth movement.

OBJECTIVE: To investigate differential expression of NOS isoforms in periodontal ligament (PDL) and bone in tension and pressure sides using a rat model of orthodontic tooth movement (OTM). MATERIALS AND METHODS: Immunohistochemistry with NOS isoform (iNOS, eNOS, and nNOS) antibodies was performed on horizontal sections of the first maxillary molars subjected to 3 and 24 hours of OTM. The PDL and adjacent osteocytes of the distopalatal root at pressure and tension areas were analyzed for expression of these proteins. The contralateral molar served as a control. Results were analyzed with one-way ANOVA and with two-way ANOVA. RESULTS: Expression of all isoforms was increased in the tension side. iNOS and nNOS expression in the pressure side with cell-free zone was decreased but in the pressure side without cell-free zone was increased. The number of eNOS-positive cells did not change, but the intensity of the staining was visibly increased in the tension side. Duration of OTM changed only the pattern of nNOS expression. Osteocyte NOS expression did not change significantly in response to OTM. CONCLUSIONS: All NOS isoforms are involved in OTM with different expression patterns between tension and pressure sides, with nNOS being more involved in early OTM events. NOS expression did not change in osteocytes, suggesting that PDL cells rather than osteocytes are the mechanosensors in early OTM events with regard to NO signaling.

Temporospatial localization of acetylcholinesterase activity in the dental epithelium during mouse tooth development.

Acetylcholinesterase (AChE), a principal modulator of cholinergic neurotransmission, also has been demonstrated to be involved in the morphogenetic processes of neuronal and non-neuronal tissues. This study shows that AChE exhibits temporospatial activity in the dental epithelium of the developing mouse tooth. To identify the AChE activity in the mouse tooth during development, we performed enzyme histochemistry on the mouse embryos from embryonic day 13 (E13) to E18 and on the incisors and molars of the neonatal mouse at 10 days after birth (P10). In the developing molars of mouse embryos, AChE activity was not found in the dental epithelium at E13 (bud stage). AChE activity first appeared in the developing cervical loops of the enamel organ at E14 (cap stage), but was not found in the enamel knot. At E18 (bell stage), AChE activity was localized in the inner enamel epithelium except the cervical-loop area. In the incisors and molars of neonatal mice (P10), AChE activity was localized in the inner enamel epithelium of the cervical-loop and enamel-free area. Overall, AChE activity was localized in the differentiating dental epithelium while the activity of butyrylcholinesterse, another cholinesterase, was located primarily in the cells of the dental follicle. The results suggest that AChE may play a role in the histo- and cytodifferentiation of dental epithelium during tooth development.

Histological evaluation of the effects of initially light and gradually increasing force on orthodontic tooth movement.

OBJECTIVE: To investigate histologically the effect of initially light and gradually increasing force on tooth movement in the rat. MATERIALS AND METHODS: Cuboids made of neodymium-iron-boron magnets (experimental groups) or titanium (control group) were bonded to the lingual surface of the right and left maxillary first molars of 18-week-old male Wistar rats. The initial distances between materials were 1.0 mm generating 4.96 gf (experimental group 1) and 1.5 mm generating 2.26 gf (experimental group 2). In three groups, rats were killed 1, 3, 7, 10, or 14 days after treatment. Histological sections were prepared and stained with hematoxylin and eosin or for tartrate-resistant acid phosphatase (TRAP) activity. The number of TRAP-positive osteoclasts was counted, and the relative hyalinized area was measured on the pressure side of periodontal ligament. RESULTS: There were significant differences in the number of osteoclasts among the three groups (P < .05). On days 1 and 3, the numbers of osteoclasts in experimental group 2 were greater than in experimental group 1. There were significant differences in the relative hyalinized area between the control group and experimental group 1 (P < .01) and between experimental groups 1 and 2 (P < .01). On days 1 and 3, the hyalinized area in experimental group 1 was larger than in experimental group 2. CONCLUSION: Initially light and gradually increasing force induced tooth movement without the lag phase and showed smooth recruitment of osteoclasts and inhibition of hyalinization.

Effect of a pulsing electromagnetic field on demineralized bone-matrix-induced bone formation in a bony defect in the premaxilla of rats.

A 2-mm non-healing bony defect was prepared in the premaxilla of male Wistar rats weighing about 180 g as a simulation of an alveolar cleft, for determination of whether a pulsing electromagnetic field (PEMF) could promote regeneration of bone induced by demineralized bone matrix (DBM). The defect was either treated with 7 mg DBM or was left as a non-grafted control. The rats were exposed to a PEMF with a frequency of 100 Hz, a 10-ms-wide burst with 100 microseconds-wide quasi-rectangular pulses, repeating at 15 Hz, and magnetic field strength of 1.5-1.8 G. Alkaline phosphatase activity increased significantly from day 7 in the DBM-graft-plus-PEMF group and from day 10 in the DBM-graft group, reaching a maximum on day 14. A greater-than-two-fold rise in alkaline phosphatase activity and a three-fold rise in the amount of 45Ca incorporation in the DBM-graft-plus-PEMF group were attained compared with those of the DBM-graft group. The DBM-graft-plus-PEMF group produced more bone with almost complete osseous bridging in the defect sites than did the group treated with DBM only on day 35. The findings indicate that PEMF had an enhancing effect on the bone-inductive properties of the DBM through the stimulation of osteoblast differentiation induced by DBM.

In situ hybridization for matrix metalloproteinase-1 and cathepsin K in rat root-resorbing tissue induced by tooth movement.

The movement of teeth during orthodontic treatment occasionally induces undesirable root resorption. Although high collagenolytic activity has been detected in resorbing tissue of deciduous teeth, the cellular origin of collagenolytic enzymes in root-resorbing tissue caused by tooth movement has not been identified. Here, rats were subject to 7 days of experimental tooth movement to induce root resorption. In situ hybridization with digoxigenin-labelled RNA probes was performed on sections of the maxillary bone to detect the mRNAs that encode matrix metalloproteinase-1 (MMP-1) and cathepsin K in root-resorbing tissue. MMP-1 mRNA was detected in fibroblastic cells, cementoblasts and osteoblasts, but not in odontoclasts nor osteoclasts. Moreover, MMP-1 mRNA was highly expressed in some cementocytes located near odontoclasts and in many osteocytes. In contrast, cathepsin K mRNA was expressed only in odontoclasts and osteoclasts. These results suggest that MMP-1 and cathepsin K are important in root resorption during tooth movement in a mode similar to bone resorption.

Expression of 72-kDa gelatinase (matrix metalloproteinase-2) in the developing mouse craniofacial complex.

Tissue remodelling is an important feature during embryogenesis. Although the matrix metalloproteinases are believed to participate in these processes, the relation between matrix metalloproteinases and tissue remodelling during craniofacial morphogenesis remains unclear. The purpose of the study was to look for the presence of enzymes involved in extracellular matrix degradation during craniofacial morphogenesis. Protein expression of the matrix metalloproteinase, 72-kDa gelatinase (matrix metalloproteinase-2, gelatinase A, 72-kDa type IV collagenase) was studied by gelatine zymography and by indirect immunofluorescence with conventional and confocal microscopy. In the anterior region of the developing mouse face, 72-kDa gelatinase was labelled mainly in the tips and peripheral regions of the nasal and facial prominences. Upon contact and fusion of the prominences, the staining was intensely localized to the zone of the fusion and the tips and peripheral regions of the nasal prominences and the maxilla. The labelling of 72-kDa gelatinase was also present in the peripheral regions of the mandible, second branchial arch, and the face around the developing eye. However, during lens vesicle formation, the staining of 72-kDa gelatinase was absent in the invaginated lens ectoderm. After the lens had completely detached from the surface ectoderm, the staining was resumed in the corneal epithelium and mesenchyme. Gelatine zymography was used to confirm the presence of active and latent 72-kDa gelatinase in the developing mouse craniofacial complex. Collectively, these data indicate that 72-kDa gelatinase may play a significant part in localized tissue remodelling during craniofacial morphogenesis and the aberrant expression or function of the enzyme could be involved in causing facial abnormalities.

Effects of insulin-like growth factor-I on the expression of osteoclasts and osteoblasts in the nasopremaxillary suture under different masticatory loading conditions in growing mice.

It is well accepted that mechanical loading inhibits bone resorption and increases in vivo bone formation. It is also known that cyclic mechanical loading, in particular, can enhance bone formation significantly. These findings suggest a significant role for mechanical stimuli in bone remodelling mediated by various local growth factors including insulin-like growth factor-I (IGF-I). Earlier studies showed that the nasal bone length and premaxillary bone width were significantly greater in mice fed a solid diet rather than a granulated diet, and that these dimensions increased significantly in a solid-diet group treated with IGF-I. The present study sought to examine the effect of IGF-I on the expression of osteoclasts and osteoblasts in the nasopremaxillary suture subjected to different masticatory loadings. For the solid-diet groups, the numbers of tartrate-resistant acid phosphatase (TRAP)-positive osteoclastic cells and osteoblasts were significantly greater in the group injected with IGF-I than in the animals injected with physiological saline. In the groups fed a granulated diet, no significant differences in the numbers of TRAP-positive osteoclastic cells and osteoblasts were found over the entire experimental period between mice injected with either IGF-I or physiological saline. It is shown that IGF-I significantly induces the expression of osteoclasts and osteoblasts and the subsequent bone remodelling, and that the effect may be additive as compared to that of mechanical masticatory loading, which seems to be more important in bone remodelling in terms of the numbers of osteoclasts and osteoblasts.

Experimental induction of palate shelf elevation in glutamate decarboxylase 67-deficient mice with cleft palate due to vertically oriented palatal shelf.

BACKGROUND: Gamma-aminobutyric acid is an inhibitory neurotransmitter, synthesized by two isoforms of glutamate decarboxylase (GAD), GAD65 and -67. Unexpectedly, inactivation of GAD67 induces cleft palate in mice. Reduction of spontaneous tongue movement resulting from decreased motor nerve activity has been related to the development of cleft palate in GAD67(-/-) fetuses. In the present study, development of cleft palate was examined histologically and manipulated with culture of the maxilla and partial resection of fetal tongue. METHODS: GAD67(-/-) mice and their littermates were used. Histological examination and immunohistochemistry were performed conventionally. Organ culture of the maxilla was carried out as reported previously. Fetuses were maintained alive under anesthesia and tips of their tongues were resected. RESULTS: Elevation of palatal shelves, the second step of palate formation, was not observed in GAD67(-/-) mice. In wild-type mice, GAD67 and gamma-aminobutyric acid were not expressed in the palatal shelves, except in the medial edge epithelium. During 2 days of culture of maxillae dissected from E13.5-E14.0 GAD67(-/-) fetuses, elevation and fusion of the palatal shelves were induced. When E13.5-15.5 mutant fetuses underwent partial tongue resection, the palatal shelves became elevated within 30 min. CONCLUSIONS: These results suggest that the potential for palate formation is maintained in the palatal shelves of GAD67(-/-) fetuses, but it is obstructed by other, probably neural, factors, resulting in cleft palate.

Cell proliferation and cell density of mesenchyme in the maxillary process and adjacent regions during facial development in the chick embryo.

Cell proliferation, as measured by DNA labeling indices was analyzed during the early development of the maxillary process. Chick embryos were labeled with [3H]thymidine for 1 h and processed for autoradiography. The percentage of labeled mesenchymal cells was determined within delineated areas in the maxillary processes and in adjacent regions. Analysis of labeling indices in each of the areas at successive stages of development demonstrated a pattern of declining rates of cell proliferation with advancing developmental age. Cell proliferation in adjacent regions declined earlier and, in some instances, faster than it did in the maxillary process. Cell density was measured in the maxillary process and the roof of the stomodeum and was found to be higher in the maxillary process throughout the period studied. Cell density and cell proliferation data were analyzed with reference to the operation of 'density-dependent inhibition' of growth as a regulatory mechanism for the observed changes. 'Density-dependent inhibition' of growth was not a satisfactory explanation for the observed differences between the maxillary process and adjacent regions.

Purification of glandular kallikrein in maxillary mucosa from humans suffering from chronic inflammation.

Glandular kallikrein was purified from human maxillary mucosa with chronic inflammation and its biochemical properties were characterized. The purification procedure consisted of extraction with 3 mol/l KCl, saturation of ammonium sulfate (66%), DEAE-Sepharose, arginine-Sepharose and Sephadex G-150 chromatographies. Maxillary mucosa with chronic inflammation contains considerable activity of glandular kallikrein, which is a serine protease with limited proteolytic activity and its biochemical properties resemble those of pancreatic kallikrein.

Characterization of soluble cyclic adenosine monophosphate-dependent protein kinase isozymes in murine embryonic palatal tissue.

Certain hormonal primary messengers identified in the mammalian palate during its ontogeny transmit information to the interior of the cell via transmembrane signaling systems that control the production of the secondary messenger cyclic adenosine monophosphate. The singular role of intracellular cyclic AMP is to activate cAMP-dependent protein kinases (cAMP-dPK). cAMP-dPK were thus identified and characterized in the developing murine embryonic palate. Incubation of cytosolic fractions of embryonic palatal tissue with cAMP resulted in a dose-dependent increase in the cAMP-dPK activity ratio. A transient elevation of basal cAMP-dPK was seen during the period of palatal ontogeny that corresponded temporally with a previously demonstrated transient elevation of palatal basal cAMP levels. Fractions of embryonic palatal tissue cytosols derived by diethylaminoethyl (DEAE)-Sephacel chromatography were analyzed for phosphotransferase activity and for [3H]-cAMP binding to the regulatory (R) subunits of cAMP-dPK. Such analyses revealed two peaks of activity on day 13 of gestation. Based on the salt concentration at which the material in these peaks eluted from DEAE, its ability to cochromatograph with authentic cAMP-dPK isozymes, its molecular weight as determined by sodium dodecyl sulfate-polycrylamide gel electrophoresis, and the ability of the material to be photoaffinity labeled with [3H]-8-azidoadenosine 3',5' cyclic phosphate, types I and II cAMP-dPK were identified. Regulatory subunits of cAMP-dPK were characterized by the binding of [3H]-cAMP to cytosolic fractions of embryonic palatal tissue. Such binding was saturable (Bmax = 1,096 fmol/mg protein) and of high affinity (Kd = 7 nM). Only cAMP and cyclic guanosine monophosphate competed in a dose-related manner with [3H]-cAMP for binding to R subunits of cAMP-dPK. Adenosine, cTMP, and adenosine triphosphate, at doses up to 10(-4) M, did not compete for binding. Temporal analysis of binding data indicated that the number of binding sites transiently decreased during day 13 of gestation. Characterization of cAMP-dPK in tissue derived from the developing mammalian palate allows consideration of cAMP-dPK as a key regulatory enzyme capable of transducing hormonally elevated intracellular levels of cAMP into metabolic responses during orofacial ontogenesis.

[Expression of matrixmetalloproteinase-8 on the bell-stage in human and rat tooth development].

OBJECTIVE: To study the expression of MMP-8 in human and rat tooth development. METHODS: Immunohistochemistry was used to detect the localization of MMP-8 protein while in situ hybridization was used to examine the expression of MMP-8 mRNA. RESULTS: The expression of MMP-8 protein was localized in odontoblast and dentin matrix at the later bell stage in human tooth germ. The dentin was denser close to the pulp cavity. The expression of MMP-8 mRNA was found in very few polarized odontoblast at the early bell stage and all polarized odontoblast at the later bell stage in rat tooth germ. CONCLUSION: The results suggested that MMP-8 involved in dentin matrix rebuilding in the process of dentin formation in human and rat dental development.

A comparative study on the occurrence and activity of extracellular matrix vesicles in young and adult rat maxillary bone.

The occurrence of matrix vesicles in the maxillary bone of normal young and adult rats was demonstrated by both ultrastructural and enzymatic studies. Transmission electron microscopy revealed that the vesicles are invested in trilaminar membranes. Occasionally, crystals were found both within the vesicles and in the surrounding matrix. Separated fractions of vesicles, membranes, and cells were studied biochemically. The amounts of vesicular protein and enzymatic activity per gram bone in the adult rats were significantly lower than in the young. This coincides with the higher number of vesicles observed in the TEM specimens of young rats when compared to that in the old ones. The specific activities of all enzymes examined in the three bone fractions obtained from both young and adult rats were similar. This indicates that no significant difference exists in the enzymatic characteristics of matrix vesicles from the maxillary bone of young and adult rats.

Isoenzyme changes during rat facial development.

A method is presented by which rat facial processes from different stages were obtained in pure fraction. The morphology, and protein and DNA contents in free dissected facial processes were determined. Facial processes of embryonic rats aged 9-15 days were analyzed by isoelectric focusing for their isoenzymic distribution of four enzymes: lactate dehydrogenase, creatine phosphokinase, fructose diphosphate aldolase and phosphoglycerate mutase. A dominance of LDH-5, LDH-4 and LDH-3 isoenzymes was observed. As a comparison, LDH isoenzymes from mandibular and maxillary processes of rat embryos aged 9-11 days only revealed LDH-5 and to a smaller extent LDH-4. The results support the presence of a prominent anaerobic metabolism in these tissues during early facial development. The change to LDH-3 development correlates well with the formation of new blood vessels. From the ninth embryonic day, isoenzyme BB of creatine phosphokinase was present and during days 10-15 MB and MM developed. Isoenzyme A4 of fructose diphosphate aldolase was present from the ninth embryonic day and isoenzymes A3C and A2C2 developed during days 10-15. From the tenth embryonic day, isoenzyme BB of phosphoglycerate mutase was present and during days 10-15 isoenzyme MB and MM developed. Isoenzyme development was first seen in mandibular processes, followed by maxillary, lateral nasal and medial nasal processes, and it preceded morphologic evidence of skeletal muscle formation.

Induction of cleft palates: effects of triamcinolone acetonide on transcription in isolated nuclei.

The effect of triamcinolone acetonide on the transcriptional activity in nuclei isolated from maternal A/J mouse livers and embryonic maxillary processes (EMP) has been studied. Triamcinolone acetonide (TAC, 13 mg/kg body weight) was administered to A/J mice on day 12.5 of gestation, and the mice were sacrificed at different time periods following injection. We find a significant increase in transcription in liver nuclei, and a decrease in this activity in nuclei from embryonic maxillary processes in response to TAC at 16 to 20 hours following injection. With the drug alpha-amanitin we show that the effect of TAC on transcription in EMP cannot be due to fluctuations in the concentration of endogenous RNA polymerase B. This is further substantiated by studies on the transcription of EMP-nuclei in the presence of exogenous DNA template. Relative to controls, the data demonstrates that the concentrations of RNA polymerases A and B in EMP-nuclei remain unchanged in response to TAC. Conversely, stimulated liver nuclei result in significant increases in the concentrations of RNA polymerases A and B. We therefore propose that in embryonic maxillary processes TAC induces changes in the chromatin template which may interfere with normal development.

In vitro activation of adenylate cyclase by norepinephrine, parathyroid hormone, calcitonin, and prostaglandins in the developing maxillary process and palatal shelf of the golden hamster.

The regional localization of hormonally sensitive adenylate cyclase within the maxillary-palatal process complex was examined in tissue homogenates at different stages during the development of the secondary palate in the golden hamster. The most potent agents capable of activating adenylate cyclase were parathyroid hormone (PTH) and calcitonin (CT). Highest activities were observed in the intact maxillary process-palatal shelf complex and the isolated maxillary process prior to and during fusion of the palate. Thereafter, neither hormone displayed a remarkable capacity to elevate enzyme activity. Parathyroid hormone and CT exhibited a similar, but considerably reduced, capacity for enzyme stimulation in the isolated palatal shelf. 1'-Norepinephrine also increased adenylate cyclase activity in both the palatal shelf and the maxillary process at the earlier stages. Prostaglandins (PG) E1, E2, and F2 alpha stimulated adenylate cyclase activity within the intact palate, the maxillary process, and the palatal shelf primarily at the earlier stages. The adenylate cyclase from the isolated palatal shelf was more sensitive to stimulation by the PGs than that from either the intact palate samples or isolated maxillary processes. The findings imply that the fusion process of the secondary palate is under a highly sensitive hormonal control mechanism.

Characterization of phosphoglycerate mutase isoenzymes from free dissected facial processes.

To characterize the three phosphoglycerate mutase (PGM) isoenzymes present in rat facial processes (types MM, BB, and MB), their sensitivity to reagents of the sulfhydryl groups and to heat treatment has been studied. Type BB PGM was not affected by the -SH group reagents; type MB PGM was inhibited about 50%, and type MM PGM was fully inhibited. Type MB PGM showed a greater heat lability than type MM PGM. There was a developmental change from type BB PGM from the 9th embryonic day to isoenzymes MB and MM on the 15th embryonic day. Isoenzyme development was first seen in mandibular processes, followed by maxillary, lateral nasal, and medial nasal processes.

A histochemical study of the development of premaxilla and maxilla during secondary palate formation in the mouse embryo.

The development of premaxilla and maxilla in the mouse fetus during secondary palate formation from the 12th to the 16th days of gestation was histochemically assessed. To determine the developmental stages, a classification based on the morphogenesis of the limbs, or the "limb score" (LS) was employed. The stage of LS coincided with the gestational age from the 13th to the 15th days. Early on the 12th day, alkaline phosphatase (ALPase) activity was intense in the mesenchyme lateral to the incisor tooth bud and latero-inferior to the inferior orbital nerve. Subsequently, osteoblasts differentiated at these two sites. The ALPase positive area grew concomitantly with the nasal capsule, the molar tooth germ, and the closure of the secondary palate. The area of bone differentiation contoured the orbital nerve and extended to the rostral part of the secondary palate. At the LS stage -6 (13.52 days), ALPase activity was observed in the mesenchyme medial to, and also surrounding the molar tooth germ. The area of osteogenesis of the secondary palate spread along the medial side of the molar tooth germ, where the formation of the medial alveolar process of the maxilla was completed by the LS stage 3 (15.35 days). The ALPase positive area extended to the horizontal palatal shelves. By late on the 16th day, the palatal process was fully developed. In parallel, bone resorption began on the molar side of the alveolar process. Acid phosphatase and tartrate-resistant acid phosphatase activities (ACPase and TRACPase activity, respectively) revealed ACPase and TRACPase positive mononuclear cells around the molar tooth germ long before ossification occurred. Our results thus suggest an involvement of the incisor tooth bud and the infra-orbital nerve in the initial osteogenesis of the premaxilla and maxilla. Enzyme activities lead to the consideration that osteoclast precursors initiate differentiation around the molar tooth germ. Ostensibly, the mechanical force from the growth of the molar tooth would promote differentiation and activation of osteoclasts located on the alveolar process. Also, the LS classification would improve and simplify future studies of the development of the secondary palate.