GRP78 promotes the osteogenic and angiogenic response in periodontal ligament stem cells.

Periodontitis is a progressive disease that ultimately leads to bone and tooth loss. A major consequence of periodontal disease is the inability to regain lost bone in the periodontium. The importance was demonstrated of glucose-regulated protein-78 (GRP78) in the osteogenic differentiation of periodontal ligament stem cells and their potential use for regeneration of the periodontium. Previous studies have shown the relationship between GRP78 and dentine matrix protein-1 (DMP1). The importance of this receptor-ligand complex in supporting the process of osteogenesis and angiogenesis was confirmed in this study. To show the function of GRP78 in mineralised tissues, transgenic periodontal ligament stem cells (PDLSCs) were generated in which GRP78 was either overexpressed or silenced. Gene expression analysis of the cells cultured under osteogenic conditions showed an increase in key osteogenic genes with the overexpression of GRP78. RNA-Seq analysis was also performed to understand the transcriptome profile associated with genotype changes. Using the database for annotation, visualisation, and integration discovery (DAVID) for the functional enrichment analysis of differentially expressed genes, the upregulation of genes promoting osteogenesis and angiogenesis with GRP78 overexpression was demonstrated. Alizarin red staining and scanning electron microscopy analysis revealed matrix mineralisation with increased calcium deposition in GRP78 overexpressing cells. The in vivo osteogenic and angiogenic function of GRP78 was shown using a subcutaneous implantation rodent model. The results suggested that GRP78 in PDLSCs can regulate the expression of both osteogenesis and angiogenesis. Therefore, GRP78 could be considered as a therapeutic target for repair of diseased periodontium.

Combined application of geranylgeranylacetone and amelogenin promotes angiogenesis and wound healing in human periodontal ligament cells.

Amelogenin directly binds to glucose-regulated protein 78 (Grp78). Cell migration activity is expected to increase when human periodontal ligament cells (hPDLCs) overexpressing Grp78 are treated with amelogenin. Geranylgeranylacetone (GGA) is a drug that induces the expression of heat shock protein and is routinely used to treat gastric ulcers. Here, we investigated the changes in the properties and behavior of hPDLCs in response to treatment with GGA and the synergistic effects of amelogenin stimulation in hPDLCs pretreated with GGA for the establishment of a novel periodontal tissue regenerative therapy. We observed that GGA treatment increased Grp78 protein expression in hPDLCs and enhanced cell migration. Microarray analysis demonstrated that increased Grp78 expression triggered the production of angiopoietin-like 4 and amphiregulin, which are involved in the enhancement of angiogenesis and subsequent wound healing via the activation of hypoxia-inducible factor 1α and peroxisome proliferator-activated receptors as well as the phosphorylation of cAMP response element-binding protein and protein kinase A. Moreover, the addition of recombinant murine amelogenin (rM180) further accelerated hPDLC migration and tube formation of human umbilical vein endothelial cells due to the upregulation of interleukin-8 (IL-8), monocyte chemotactic protein 1, and IL-6, which are also known as angiogenesis-inducing factors. These findings suggest that the application of GGA to gingival tissue and alveolar bone damaged by periodontal disease would facilitate the wound healing process by inducing periodontal ligament cells to migrate to the root surface and release cytokines involved in tissue repair. Additionally, supplementation with amelogenin synergistically enhanced the migratory capacity of these cells while actively promoting angiogenesis. Therefore, the combined application of GGA and amelogenin may establish a suitable environment for periodontal wound healing and further drive the development of novel therapeutics for periodontal tissue regeneration.

Nonuniformity in ligaments is a structural strategy for optimizing functionality.

Ligaments serve as compliant connectors between hard tissues. In that role, they function under various load regimes and directions. The 3D structure of ligaments is considered to form as a uniform entity that changes due to function. The periodontal ligament (PDL) connects the tooth to the bone and sustains different types of loads in various directions. Using the PDL as a model, employing a fabricated motorized setup in a microCT, we demonstrate that the fibrous network structure within the PDL is not uniform, even before the tooth becomes functional. Utilizing morphological automated segmentation methods, directionality analysis, as well as second harmonic generation imaging, we find high correlation between blood vessel distribution and fiber density. We also show a structural feature in a form of a dense collar around the neck of the tooth as well as a preferred direction of the fibrous network. Finally, we show that the PDL develops as a nonuniform structure, with an architecture designed to sustain specific types of load in designated areas. Based on these findings, we propose that ligaments in general should be regarded as nonuniform entities, structured already at developmental stages for optimal functioning under variable load regimes.

PDLSCs Regulate Angiogenesis of Periodontal Ligaments via VEGF Transferred by Exosomes in Periodontitis.

Abnormal angiogenesis is one of the significant features in periodontitis leading to progressive inflammation, but angiogenic changes of periodontal ligaments under inflammatory condition were rarely reported. Periodontal ligament stem cells (PDLSCs) were a kind of dental stem cells associated with vascularization. Here we investigated the alteration of angiogenesis of periodontal ligament in periodontitis, and revealed an exosome-mediated pathway to support the effect of PDLSCs on angiogenic improvement. Vascular specific marker CD31 and VEGFA were found to be highly expressed in periodontal ligaments of periodontitis. The VEGFA expression was up-regulated in inflamed PDLSCs compared to control, meanwhile the tube formation of HUVECs was improved when co-cultured with inflamed PDLSCs. Exosomes secretion of PDSLCs was augmented by inflammation, and promoted angiogenesis of HUVECs, whereas blocking secretion of exosomes led to degenerated angiogenesis of HUVECs. Exosome-trasferred VEGFA was proven to be the crucial communicator between PDLSCs and HUVECs. Inflammation inhibited miR-17-5p expression of PDLSCs and relieved its target VEGFA. However, overexpression of miR-17-5p blocked the pro-angiogenic ability of inflamed PDLSCs. In conclusion, the findings indicated that vascularization of periodontal ligaments was enhanced, and inflammatory micro-environment of periodontitis facilitated pro-angiogenesis of PDLSCs through regulating exosome-mediated transfer of VEGFA, which was targeted by miR-17-5p.

Microanatomical changes and biomolecular expression at the PDL-entheses during experimental tooth movement.

The novel aspect of this study was to contextualize the co-localization of biomolecular expression in widened and narrowed periodontal ligament (PDL)-space within a mechanically activated periodontal complex. The PDL is unique as it is the only ligament with both innervation and vascularization. Maxillary molars in 6-week-old male C57BL/6 mice (N = 5) were experimentally translated for 2 weeks using an elastic spacer. Contralateral teeth were used as controls. Mechanical testing of the periodontal complex of a mouse in situ and imaging using X-ray micro-computed tomography (micro-XCT) illustrated deformations within blood vessels (BV) of the PDL. PDL-bone and PDL-cementum entheses at the widened and narrowed PDL-spaces following experimental tooth movement (ETM) illustrated osterix (OSX), bone sialoprotein (BSP), cluster of differentiation 146 (CD146), and protein gene product 9.5 (PGP9.5), indicating active remodeling at these sites. PGP9.5 positive nerve bundles (NBs) were co-localized with multinucleated cells (MCs), Howship's resorption lacunae, and CD146 positive BVs. Association between nerves and MC was complemented by visualizing the proximity of osmium tetroxide stained NBs with the ultrastructure of MCs by performing scanning transmission electron microscopy. Spatial association of NB with BV, and NB with MC, provided insights into the plausible co-activation of NBs to initiate osteoclastic activity. Resorption of mineral occurred as an attempt to restore PDL-space of the load-bearing complex, specifically at the PDL-entheses. Mapping of anatomy-specific structural elements and their association with regenerative molecules by correlating light and electron micrographs provided insights into the use of these extracellular matrix molecules as plausible targets for pharmacological interventions related to tooth movement. Within the realm of tissue regeneration, modulation of load can reverse naturally occurring mineral formation to experimentally induced resorption, and naturally occurring mineral resorption to experimentally induced formation at the enthesial sites to permit tooth translation.

Green tea prevents vascular disturbs and attenuates periodontal breakdown in long-term hyperglycaemia in T1D rats.

AIM: The effects of green tea on the modulation of vascularization during the progression of spontaneous periodontitis in long-term hyperglycaemia in streptozotocin-induced type 1 diabetic (T1D) rats were evaluated. MATERIALS AND METHODS: Wistar rats normoglycaemic (NG) and T1D were divided into two control groups, which received water (NG-W and T1D-W) and two experimental groups that received green tea (NG-GT and T1D-GT). Periodontal structures were evaluated by microtomographic and histological analyses. Number of immunostained cells for VEGF (NcVEGF+/mm2 ) and CD31 (NcCD31+/mm2 ), as well microvessel density (MVD) in the periodontal ligament (PDL) were evaluated. RESULTS: Long-term hyperglycaemia in T1D-W rats induced vascular alterations in PDL with a reduction of 36% in MVD, a decrease of 33% in NcCD31+/mm2 and an increase of 53% in NcVEGF+/mm2 . Concomitantly, a severe degree of periodontitis with higher reduction in bone volume and periodontal bone level was observed. In T1D-GT, green tea maintained the MVD, NcCD31+/mm2 and NcVEGF+/mm2 in the PDL similar to normoglycaemic groups. Clinically, in T1D-GT rats, green tea reduced dental plaque accumulation and the degree of periodontitis when compared to T1D-W. CONCLUSION: Daily green tea consumption has a therapeutic effect on the diabetic vascular disorder in PDL and the progression of periodontitis in long-term hyperglycaemia in T1D rats.

[Morphological evaluation of singlet phototherapy in the treatment of periodontal diseases in an experimental study]. / Morfologicheskaia otsenka singletnoi fotooksiterapii pri lechenii zabolevanii parodonta v éksperimental'nom issledovanii.

The aim of the study was to compare the effectiveness of laser singlet phototherapy and traditional photodynamic therapy the treatment of periodontal diseases in an animal model. The experimental model involved 70 male rats Wistar in which periodontitis was modeled and treated: in group I (30 animals) a nanosecond laser device for medical use with a wavelength of 1270 nm was used for 7 sessions in a 400 ns pulse mode, an average radiation power of 2 W, and a radiation density of 200 J/cm2, group II (30 animals) received photodynamic therapy with the administration of a photosensitizer, followed by irradiation with a laser wavelength of 660 nm for 7 sessions 2 Wt average radiation power, group III (controls, 10 animals) - traditional drug therapy. Morphological studies were performed on 7, 14 and 21 day after treatment. On day 7th and 14th the study revealed In group I the presence of full blood vessels and diffuse expressed leukocyte infiltration with an admixture of macrophages, in group II - pronounced edema of the tissue and vasoconstriction. On day 21 the picture included in group I regenerated periodontal ligament with dilated full blood vessels on the border with the bone beams of the alveolar bone, in group II a moderately pronounced edema of the periodontal ligament with single dilated vessels, in controls significantly destroyed periodontal ligament substituted with granulation tissue and periodontal ligament. Thus, the treatment of periodontitis with the methods of singlet phototherapy leads to the development of reactive inflammation and significant vascularization of periodontal tissues which contributes to the rapid regeneration and stability of remission.

Electrical stimulation enhances tissue reorganization during orthodontic tooth movement in rats.

OBJECTIVE: This study evaluated the effects of a low-intensity electric current on tissue reorganization during experimental orthodontic tooth movement. MATERIALS AND METHODS: Thirty-two animals were divided into two groups evaluated on days 3 and 7: OTM-orthodontic tooth movement and OTM + MC-orthodontic tooth movement and microcurrent application (10 µA/5 min). The samples were processed for histological, morphometric, and Western blotting analysis. RESULTS: Analysis of the periodontal ligament (PL) showed a significantly smaller number of granulocytes in the OTM + MC group on day 7.The number of fibroblasts was significantly higher in the OTM + MC group on days 3 and 7. The area of birefringent collagen fibers was more organized in the OTM + MC group on days 3 and 7. The number of blood vessels was significantly higher in the OTM + MC group on day 7. Microcurrent application significantly increased the number of osteoclasts in the compression region of the PL. In the OTM + MC group on day 7 of tooth movement, the expression of TGF-ß1 and VEGF was significantly reduced whereas the expression of bFGF was increased in PL. CONCLUSIONS: Electrical stimulation enhances tissue responses, reducing the number of granulocytes and increasing the number of fibroblasts, blood vessels, and osteoclasts and modulates the expression of TGF-ß1, VEFG, and bFGF. CLINICAL RELEVANCE: This technique is used in many areas of medicine, but poorly explored in dentistry and orthodontics. This treatment is cheap and non-invasive and can be applied by own orthodontist, and it can improve the treatment with a faster and safe tooth movement, without pain.

A qualitative investigation of RANKL, RANK and OPG in a rat model of transient ankylosis.

INTRODUCTION: Previous studies have found ankylosis occurs as a part of the inflammatory process of aseptic root resorption initiated in a rat model. The physiologic mechanisms behind the development of dentoalveolar ankylosis and healing response are still unclear. While receptor activator of nuclear factor-κß ligand (RANKL), receptor activator of nuclear factor-κß (RANK) and osteoprotegerin (OPG) have gained momentum in the understanding of resorption, no study to date has investigated their role in dentoalveolar ankylosis. AIMS: The aims of this study were to investigate if, and when, ankylosis occurred in the rat PDL, whether the resolution of ankylosis occurred with time and, finally, to observe the expression of RANKL, RANK and OPG during the ankylotic process. MATERIALS AND METHODS: Dry ice was applied for 20 minutes to the upper right first molar crown of 15 eight-week-old, male Sprague-Dawley rats. An additional three rats served as untreated external controls. Groups of three rats were sacrificed after the thermal insult on day 0, 4, 7, 14 and 28 respectively. Each maxilla was dissected out and processed for histological examination and RANKL, OPG and RANK immunohistochemistry. RESULTS: By the use of light microscopy and H&E staining, no ankylosis was detected in the external control group and the experimental groups at days 0 and 4. On day 7, disruption within the periodontal ligament was observed in the interradicular region and the initial signs of ankylosis were seen in the form of finger-like projections extending from the alveolar bone towards the cementum. Fourteen days after the thermal insult, all animals exhibited extensive ankylosis that spanned the entire interradicular periodontal space. At 28 days, the development of ankylosis appeared to have ceased and repair was observed, together with an intact periodontal ligament in all but one rat. Positive staining results were obtained with RANKL, RANK and OPG antibodies. The expressions of RANKL, RANK and OPG were similar in the external control group, 0-, 4-, and 28-day experimental groups. In the 7- and 14-day experimental groups, RANKL, RANK and OPG were expressed in the blood vessels within the ankylotic regions. CONCLUSIONS: During the development of ankylosis and its resolution, it was concluded from their simultaneous presence that there is a complex interaction between RANKL, RANK and OPG that requires further investigation.

The inter-relation between epithelial cells of Malassez and vessels studied immunohistochemically in the periodontal membrane of human primary and permanent teeth.

BACKGROUND: Only few immunohistochemical studies have focused on the periodontal membrane in human primary teeth. Recently, studies on epithelial cells of Malassez and innervation have been published. Studies on the inter-relation between vessels and the epithelial cells of Malassez are seemingly lacking. AIM: he aim of this immunohistochemical study is to describe the histological inter-relation between epithelial cells of Malassez and vessels in the periodontal membrane close to the root surface of human primary and permanent teeth. METHODS: Twenty-nine human primary teeth and 15 permanent teeth were extracted in connection with dental treatment. The teeth were fixated, embedded in paraffin, cut in serial sections and examined immunohistochemically for epithelial cells of Malassez using wide spectrum screening and vessels using Von Willebrand Factor VIII. RESULTS: The study showed that vessels and epithelial cells of Malassez are seen parallel to the root surface. The vessels are seen on that side of the epithelial cells of Malassez, which are not facing the root surface. CONCLUSION: The vascularization appeared similar in primary and permanent teeth.

[Orthodontic tooth movement and expression of calcium regulating hormone].

Nociception by orthodontic tooth movement stimulate Trigeminal nerve free endings in periodontal ligament (PDL) , and neuropeptides such as substance P and CGRP are synthesized in Trigeminal ganglion sensory cells and released both centrally and peripherally around blood vessels in PDL and pulp. Neuropeptides such as CGRP and substance P are the signal transmitter of pain and might modulate vascular enlargement, blood flow or vascular permeability. CGRP receptor for its subunit, receptor activity modifying protein 1 (RAMP 1) distributed on osteoclasts and osteoblasts in PDL. CGRP may have effects on bone remodeling due to not only inhibiting bone resorption like calcitonin but also directly stimulating bone formation in the luxated PDL and during experimental tooth movement.

Supraperiosteal transport distraction osteogenesis for reconstructing a segmental defect of the mandible.

Distraction osteogenesis using the maximal osteogenic potential of the periosteum is introduced and demonstrated in 2 clinical cases. This technique includes minimizing cuts to and reflection of the periosteum on a transport segment; repositioning and suturing the cut periosteum to cover an osteotomy line, which becomes the distraction gap; and fixing an internal distraction device supraperiosteally. The cases involve reconstruction of a segmental defect of the mandible due to a recurrent ameloblastoma. A 51 + 58-mm posterior body and ramus and a 41 + 33-mm anterior body are reconstructed through 2-step and bilateral distraction osteogenesis, respectively. These cases proved the feasibility of the concept of the supraperiosteal transport distraction osteogenesis.

Histomorphological study of myelinated nerve fibres in the periodontal ligament of human canine.

OBJECTIVE: This study aims to describe the human periodontal ligament (PDL) using serial sections, with a focus on mechanoreceptor distribution and morphology. MATERIALS AND METHODS: One permanent lower canine with surrounding PDL and alveolar bone tissues was retrieved from a human cadaver. After being embedded into paraffin block, the canine was horizontally cut in 6 µm thick serial sections. At root levels of 0.3, 1.5, 3, 4.5 and 6 mm from the apex, five slices each level were evaluated. Immunocytochemisty was performed on the same serial sections, enabling a more reliable description of neural structures. RESULTS: The distribution of myelinated fibres varied from apical to coronal level, with a total number of 38 at 0.3 mm from the apex, 25 at 1.5 mm, 25 at 3 mm, 31 at 4.5 mm and 32 at 6 mm. At all times, mesial and buccal regions were typically more densely innervated (p < 0.01) except at the 3 mm level. The average density of myelinated nerve fibres increased by arriving closer to the apex. However, the average diameter did not show any significant differences amongst quadrants or root levels (p > 0.05). The average diameter of myelinated fibres varied between 5.3-7.8 µm. Grouped myelinated axons were twice as common as isolated ones, with the innervation being rather close to the alveolar bone. Isolated myelinated axons showed a tendency to group around large blood vessels. CONCLUSION: The present results add to the understanding of human PDL innervation, indicating dense innervations by myelinated nerve fibres in close proximity to collagen fibres and alveolar bone. It also reveals that apical as well as mesial and buccal sites of the human canine are more densely innervated.

Three-dimensional morphometry of strained bovine periodontal ligament using synchrotron radiation-based tomography.

The periodontal ligament (PDL) is a highly vascularized soft connective tissue. Previous studies suggest that the viscous component of the mechanical response may be explained by the deformation-induced collapse and expansion of internal voids (i.e. chiefly blood vessels) interacting with liquids (i.e. blood and interstitial fluids) flowing through the pores. In the present work we propose a methodology by means of which the morphology of the PDL vascular plexus can be monitored at different levels of compressive and tensile strains. To this end, 4-mm-diameter cylindrical specimens, comprising layers of bone, PDL and dentin covered by cementum, were strained at stretch ratios ranging from lambda = 0.6 to lambda = 1.4 and scanned using synchrotron radiation-based computer tomography. It was concluded that: (1) the PDL vascular network is layered in two distinct planes of blood vessels (BVs): an inner layer (close to the tooth), in which the BVs run in apico-coronal direction, and an outer layer (close to the alveolar bone), in which the BVs distribution is more diffuse; (2) during tension and compression, the porosity tissue is kept fairly constant; (3) mechanical straining induces important changes in BV diameters, possibly modifying the permeability of the PDL and thus contributing to the viscous component of the viscoelastic response observed under compressive forces.

Immunohistochemical localization of elastin, fibrillins and microfibril-associated glycoprotein-1 in the developing periodontal ligament of the rat molar.

BACKGROUND AND OBJECTIVE: Elastic system fibers are a major component of the periodontal ligament, but little information is available about their detailed composition or the mechanism of elastogenesis in the developing periodontal ligament. The purpose of this study was to investigate immunolocalization of elastin, fibrillins and microfibril-associated glycoprotein-1 (MAGP-1) in the developing periodontal ligament of the rat molar. MATERIAL AND METHODS: Frozen sections of demineralized as well as non-demineralized periodontal ligament of Wistar rats of various ages from 19 days to 7 weeks were incubated with anti-elastin, anti-fibrillin-1 and -2 and anti-MAGP-1 antibodies followed by peroxidase-conjugated secondary antibodies. After incubation with diaminobenzidine solution, immunoreaction products were observed with a light microscope. RESULTS: In the developing periodontal ligament of 19-day-old rats, fibers immunopositive to elastin were not present, but fibers positively stained for fibrillin-2 and MAGP-1 were widely distributed throughout the ligament. The latter fibers were arranged in the apico-occlusal direction along with blood vessels. In 3-week-old rats, fibers stained for elastin were observed for the first time in the apical region of the ligament. The number and distribution pattern of these elastin-positive fibers was basically the same as those in rats aged 5 and 7 weeks. In contrast, fibrillin-2- and MAGP-1-positive fibers were more extensively distributed in the ligament, and their pattern of distribution was comparable to that of reported oxytalan fibers. Fibrillin-1 was, however, not detected either in demineralized sections or in non-demineralized sections, indicating its absence in periodontal ligament. CONCLUSION: Elastin expressed in the periodontal ligament assembled into elaunin fibers in the vicinity of blood vessels. Both fibrillin-2 and MAGP-1 are structural components not only of the elastin-associated microfibrils but also of elastin-free microfibrils, with possible roles in elastogenesis and in periodontal ligament homeostasis.

Development of tissue-engineered human periodontal ligament constructs with intrinsic angiogenic potential.

One approach to treat periodontal diseases is grafting of tissue-engineered periodontal ligaments. Therefore, periodontal ligaments were constructed by layering cell sheets. A cell sheet was prepared by enzymatic digestion of salmon collagen gel on which human periodontal ligament fibroblasts (HPLFs) were co-cultured with or without human umbilical vein endothelial cells (HUVECs). Three cell sheets were layered and then cultured in angiogenic media, in which the HUVECs were found to form capillary-like structures when co-cultured on the HPLFs. The layered HPLFs sheets with HUVEC co-culture (PL-EC construct) demonstrated longer survival, higher alkaline phosphatase activities and lower osteocalcin production than layered HPLFs sheets without HUVEC co-culture (PL construct). Hematoxylin-eosin and Masson's trichrome staining of histological sections showed that cell density, mass and extracellular matrix deposition of the PL-EC construct were higher than those of the PL construct. Furthermore, CD31 immunostaining revealed the formation of capillary-like structures throughout the PL-EC construct. In conclusion, we successfully developed tissue-engineered periodontal ligament constructs with intrinsic angiogenic potential using cell sheet engineering and HUVEC co-culture.

The constitutive activation of extracellular signal-regulated kinase 1 and 2 in periodontal ligament nerve fibers.

BACKGROUND: The extracellular signal-regulated kinases 1 and 2 (ERK1/2) have been implicated in the inflammation-dependent sensitization of nociceptors. Because the periodontal ligament (PDL) contains numerous nociceptors and mechanoceptors, phosphorylation of ERK1/2 was investigated in nerve fibers of the PDL to elucidate the role of constitutive local activation of ERK1/2 in peripheral sensitization. METHODS: Decalcified free-floating sections of rat molars with PDL were incubated using total (t)-ERK1/2 and phosphorylated (p)-ERK1/2 antibodies. For identification of nerve fibers in the PDL, double staining was performed using protein gene product 9.5 (PGP 9.5) with p-ERK1/2. To test whether p-ERK1/2 activated in sensory and mechanoreceptive terminals, double incubations were performed using p-ERK1/2 with calcitonin gene-related peptide (CGRP) and with calretinin. Labeled nerve fibers were quantified by the point-counting method. RESULTS: In cervical, midroot, and apical zones of the PDL, t-ERK1/2- and p-ERK1/2-labeled nerve fibers were found in close association with blood vessels. The p-ERK1/2-labeled free nerve fibers were often detected in cervical and apical areas of the PDL. In nerve fibers of the PDL, p-ERK1/2 was colocalized with PGP 9.5, CGRP, and calretinin. CONCLUSIONS: The perivascular distribution of t-ERK1/2 and p-ERK1/2 in nerve fibers in the PDL is compatible with a role for the constitutive activation of ERK1/2 in the neural regulation of blood vessels in the PDL. The colocalizations of p-ERK1/2 with CGRP and calretinin indicate that ERK1/2 is constitutively activated in a subpopulation of sensory and mechanoreceptive nerve terminals in the PDL.

Morphological changes in the rat periodontal ligament and its vascularity after experimental tooth movement using superelastic forces.

The aim of this study was to statistically assess the morphological changes of the rat periodontal ligament (PDL) and its vascularity in relation to varied magnitudes of superelastic force in experimental tooth movement using nickel-titanium (NiTi) alloy wire. Forces of 0.8, 1.6, 4, 8, and 18 g were applied to the upper first molars of five groups of 10-week-old male Wistar rats (300-320 g) for 1, 7, 14, 21, and 28 days. A control group with no orthodontic appliance application was assessed in accordance with the five experimental periods. The specimens were observed under light microscopy, processed by computer imaging, and analysed statistically with Tukey's HSD non-parametric test. One day after the start of the experiment, a few blood vessels could be seen in the compressed PDL with forces of 0.8 and 1.6 g. The cross-sectional areas of blood vessels (CAV) and periodontal ligament (CAPL) in the experimental groups where a force of over 4 g was applied were significantly smaller than those where 0.8 and 1.6 g forces were used, and in the control group. On day 7, large CAV were seen in the 1.6, 4, and 8 g groups. On day 28, the 8 and 18 g groups showed significantly larger CAPL than the 0.8, 4 g, or control groups. The findings suggest that a light continuous force, under 1.6 g, maintains the vascular structure during experimental tooth movement. In contrast, a heavy continuous force over 4 g causes the vascular structure to be absent in the early stages of tooth movement, but a dynamic regeneration of the PDL with vascularity and expansion follows.

Regional structural characteristics of bovine periodontal ligament samples and their suitability for biomechanical tests.

Mechanical testing of the periodontal ligament requires a practical experimental model. Bovine teeth are advantageous in terms of size and availability, but information is lacking as to the anatomy and histology of their periodontium. The aim of this study, therefore, was to characterize the anatomy and histology of the attachment apparatus in fully erupted bovine mandibular first molars. A total of 13 teeth were processed for the production of undecalcified ground sections and decalcified semi-thin sections, for NaOH maceration, and for polarized light microscopy. Histomorphometric measurements relevant to the mechanical behavior of the periodontal ligament included width, number, size and area fraction of blood vessels and fractal analysis of the two hard-soft tissue interfaces. The histological and histomorphometric analyses were performed at four different root depths and at six circumferential locations around the distal and mesial roots. The variety of techniques applied provided a comprehensive view of the tissue architecture of the bovine periodontal ligament. Marked regional variations were observed in width, surface geometry of the two bordering hard tissues (cementum and alveolar bone), structural organization of the principal periodontal ligament connective tissue fibers, size, number and numerical density of blood vessels in the periodontal ligament. No predictable pattern was observed, except for a statistically significant increase in the area fraction of blood vessels from apical to coronal. The periodontal ligament width was up to three times wider in bovine teeth than in human teeth. The fractal analyses were in agreement with the histological observations showing frequent signs of remodeling activity in the alveolar bone - a finding which may be related to the magnitude and direction of occlusal forces in ruminants. Although samples from the apical root portion are not suitable for biomechanical testing, all other levels in the buccal and lingual aspects of the mesial and distal roots may be considered. The bucco-mesial aspect of the distal root appears to be the most suitable location.

Effect of additional surgical procedures on root development of transplanted immature third molars.

The aim of this investigation was to determine the influence of additional surgical procedures on the root development of transplanted teeth. The study sample consisted of 90 immature third molars transplanted in 88 patients. All transplanted teeth were at root development stages 3 to 4. Free bone autografts were used in 23 cases (bone autograft group), mainly because of vertical atrophy of the alveolar process. A splitting osteotomy of the alveolar process was performed in 25 cases with marked horizontal atrophy (osteotomy group). Forty-two teeth transplanted into a fresh extraction site immediately after extraction of the non-retainable tooth served as controls. At root development stage 3, significant differences were determined between the osteotomy and the control groups in final root length (P<0.001) and root length increment (P=0.004). Transplants in the osteotomy group revealed a significantly lower root length increment than transplants in the bone autograft group (P=0.008). No significant intergroup differences were observed at root development stage 4. These results indicate that a splitting osteotomy of the alveolar process has a negative effect on root development of transplanted teeth at earlier developmental stages.