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.

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.

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.

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.

Periodontal Blood Flow Protects the Alveolar Bone from Thermal Injury during Thermoplasticized Obturation: A Finite Element Analysis Study.

INTRODUCTION: The purpose of this study was to investigate the heat transfer during thermoplastic obturation and the cooling capacity of blood flow in the periodontal ligament (PDL) using finite element analysis (FEA). METHODS: A 3-dimensional digital tooth model was constructed based on micro-computed tomographic scanning of a mandibular first molar after chemomechanical preparation in vitro. A layer of PDL with or without simulated blood flow was built on the root portion of the tooth in software. Two heat-assisted obturation techniques (ie, the single-wave condensation technique and the warm vertical compaction technique followed by backfilling with injectable gutta-percha) were examined using an FEA package. RESULTS: In the model without blood flow, the highest temperature at the alveolar bony aspect of the PDL was 50.0°C along the distal canal and 52.5°C for the mesiolingual canal when the single-wave technique was used. With the warm vertical compaction technique, the highest temperature was 47.3°C for the distal canal and 47.8°C for the mesiolingual canal. In the model with simulated periodontal blood flow, a notable drop in the peak temperatures at the root surface and at the adjoining alveolar bone was observed for both the distal and mesiolingual canals; all peak temperatures at the PDL fell below 47°C regardless of the obturation techniques used. The greatest rise in temperature was situated at the furcation aspect of the middle third of both roots. CONCLUSIONS: The cooling capacity of blood flow in the PDL is a factor that must be considered in the investigation of heat transfer during thermoplastic obturation.

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.

Mechanobiology of the tooth movement during the orthodontic treatment: a literature review.

Orthodontic tooth movement differs significantly from the physiological tooth movement, as it determines a biological response of the surrounding tissues of the teeth, resulting in a remodelling of the periodontal ligament and the alveolar bone. The result is a biochemical adaptive response to the application of the orthodontic force with the reorganization of the intracellular and the extracellular matrix, in addition to a change of the local vascularization. This in turn leads to the synthesis and the release of arachidonic acid, growth factors, metabolites, cytokines and various enzymes. Biologically, not only the intensity of the force, but also its duration and the tissue response to the application of the same are important for tooth movement. Having these insights it will possible to examine the concept of optimal orthodontic force, a determining factor for the success of orthodontic treatment. The purpose of this revision was to describe the biological processes and future perspective of the application of orthodontic force, by providing relevant information to understand the changes at the molecular and cellular level occurring when the tissues are subjected to such forces. Knowledge on the subject of mechanics and biology in orthodontics is constantly growing, producing an increasingly strong basis for clinical success.

Action Mechanism of Fibroblast Growth Factor-2 (FGF-2) in the Promotion of Periodontal Regeneration in Beagle Dogs.

Fibroblast growth factor-2 (FGF-2) enhances the formation of new alveolar bone, cementum, and periodontal ligament (PDL) in periodontal defect models. However, the mechanism through which FGF-2 acts in periodontal regeneration in vivo has not been fully clarified yet. To reveal the action mechanism, the formation of regenerated tissue and gene expression at the early phase were analyzed in a beagle dog 3-wall periodontal defect model. FGF-2 (0.3%) or the vehicle (hydroxypropyl cellulose) only were topically applied to the defect in FGF-2 and control groups, respectively. Then, the amount of regenerated tissues and the number of proliferating cells at 3, 7, 14, and 28 days and the number of blood vessels at 7 days were quantitated histologically. Additionally, the expression of osteogenic genes in the regenerated tissue was evaluated by real-time PCR at 7 and 14 days. Compared with the control, cell proliferation around the existing bone and PDL, connective tissue formation on the root surface, and new bone formation in the defect at 7 days were significantly promoted by FGF-2. Additionally, the number of blood vessels at 7 days was increased by FGF-2 treatment. At 28 days, new cementum and PDL were extended by FGF-2. Moreover, FGF-2 increased the expression of bone morphogenetic protein 2 (BMP-2) and osteoblast differentiation markers (osterix, alkaline phosphatase, and osteocalcin) in the regenerated tissue. We revealed the facilitatory mechanisms of FGF-2 in periodontal regeneration in vivo. First, the proliferation of fibroblastic cells derived from bone marrow and PDL was accelerated and enhanced by FGF-2. Second, angiogenesis was enhanced by FGF-2 treatment. Finally, osteoblastic differentiation and bone formation, at least in part due to BMP-2 production, were rapidly induced by FGF-2. Therefore, these multifaceted effects of FGF-2 promote new tissue formation at the early regeneration phase, leading to enhanced formation of new bone, cementum, and PDL.

Nicotine effect on bone remodeling during orthodontic tooth movement: histological study in rats.

INTRODUCTION: Nicotine is harmful to angiogenesis, osteogenesis and synthesis of collagen. OBJECTIVE: The aim of this study was to investigate the effect of nicotine on bone remodeling during orthodontic movement in rats. METHODS: Eighty male Wistar rats were randomly divided into three groups: Group C (control), group CM (with orthodontic movement) and group NM (nicotine with orthodontic movement) groups. The animals comprising groups C and CM received 0.9% saline solution while group NM received nicotine solution (2 mg/kg). A nickel-titanium closed-coil spring was used to induce tooth movement. The animals were euthanized and tissue specimens were histologically processed. Blood vessels, Howship's lacunae and osteoclast-like cells present in the tension and compression areas of periodontal ligaments were quantified. The extent of bone formation was evaluated under polarized light, to determine the percentage of immature/mature collagen. RESULTS: It was observed lower blood vessel densities in the NM group in comparison to the CM group, three (p < 0.001) and seven (p < 0.05) days after force application. Osteoclast-like cells and Howship's lacunae in the NM group presented lower levels of expression, in comparison to the CM group, with significant differences on day 7 (p < 0.05 for both variables) and day 14 (p < 0.05 for osteoclast-like cells and p < 0.01 for Howship's lacunae). The percentage of immature collagen was increased in the NM group in comparison to the CM group, with a statistically significant difference on day 3 (p < 0.05), day 7 (p < 0.001), day 14 (p < 0.001) and day 21 (p < 0.001). CONCLUSIONS: Nicotine affects bone remodeling during orthodontic movement, reducing angiogenesis, osteoclast-like cells and Howship's lacunae, thereby delaying the collagen maturation process in new bone matrix.

Nicotine effect on bone remodeling during orthodontic tooth movement: Histological study in rats

Introduction: Nicotine is harmful to angiogenesis, osteogenesis and synthesis of collagen. Objective: The aim of this study was to investigate the effect of nicotine on bone remodeling during orthodontic movement in rats. Methods: Eighty male Wistar rats were randomly divided into three groups: Group C (control), group CM (with orthodontic movement) and group NM (nicotine with orthodontic movement) groups. The animals comprising groups C and CM received 0.9% saline solution while group NM received nicotine solution (2 mg/kg). A nickel-titanium closed-coil spring was used to induce tooth movement. The animals were euthanized and tissue specimens were processed histologically. We quantified blood vessels, Howship's lacunae and osteoclast-like cells present in the tension and compression areas of periodontal ligaments. The extent of bone formation was evaluated under polarized light to determine the percentage of immature/mature collagen. Results: We observed lower blood vessel densities in the NM group in comparison to the CM group, three (p < 0.001) and seven (p < 0.05) days after force application. Osteoclast-like cells and Howship's lacunae in the NM group presented lower levels of expression in comparison to the CM group, with significant differences on day 7 (p < 0.05 for both variables) and day 14 (p < 0.05 for osteoclast-like cells and p < 0.01 for Howship's lacunae). The percentage of immature collagen increased in the NM group in comparison to the CM group with a statistically significant difference on day 3 (p < 0.05), day 7 (p < 0.001), day 14 (p < 0.001) and day 21 (p < 0.001). Conclusions: Nicotine affects bone remodeling during orthodontic movement, reducing angiogenesis, osteoclast-like cells and Howship's lacunae, thereby delaying the collagen maturation process in developed bone matrix. .

Introdução: a nicotina apresenta efeito prejudicial sobre a angiogênese, osteogênese e síntese de colágeno. Objetivo: investigar a ação da nicotina sobre a remodelação óssea durante o movimento dentário induzido em ratos. Métodos: oitenta ratos machos Wistar foram divididos em três grupos: grupo C (sem indução de movimento dentário e sem a ação da nicotina - controle); grupo CM (indução de movimento dentário) e grupo NM (indução de movimento dentário associado à ação da nicotina). Os animais dos grupos C e CM receberam solução salina a 0,9% e os animais do grupo NM receberam nicotina (solução PA a 98% diluída em solução salina a 0,9% estéril) por via subcutânea (2mg/kg). Após a eutanásia dos animais, com 3, 7, 14 e 21 dias de uso da mola ortodôntica, os espécimes teciduais foram processados histologicamente e quantificou-se o número de vasos sanguíneos, lacunas de Howship e células osteoclásticas nos lados de tração e compressão do ligamento periodontal. A neoformação óssea foi avaliada por meio de luz polarizada, para determinar a porcentagem de colágeno maduro e imaturo. Resultados: observou-se que a quantidade de vasos sanguíneos diminuiu no grupo NM, quando comparado ao grupo CM, nos períodos de três (p < 0,001) e sete (p < 0,05) dias. Quanto às células osteoclásticas e lacunas de Howship, o grupo NM apresentou menores níveis de expressão em relação ao grupo CM, com diferença estatisticamente significativa nos períodos de 7 e 14 dias. A porcentagem de colágeno imaturo apresentou-se aumentada no grupo NM, quando comparado ao grupo CM, em todos os períodos analisados, com diferença e...

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.

In vitro analysis of human periodontal microvascular endothelial cells.

BACKGROUND: Endothelial cells (ECs) participate in key aspects of vascular biology, such as maintenance of capillary permeability, initiation of coagulation, and regulation of inflammation. According to previous reports, ECs have revealed highly specific characteristics depending on the organs and tissues. However, some reports have described the characteristics of the capillaries formed by human periodontal ECs. Therefore, the aim of the present study is to examine the functional characteristics of the periodontal microvascular ECs in vitro. METHODS: Human periodontal ligament-endothelial cells (HPDL-ECs) and human gingiva-endothelial cells (HG-ECs) were isolated by immunoprecipitation with magnetic beads conjugated to a monoclonal anti-CD31 antibody. The isolated HPDL-ECs and HG-ECs were characterized to definitively demonstrate that these cell cultures represented pure ECs. Human umbilical-vein ECs and human dermal microvascular ECs were used for comparison. These cells were compared according to the proliferation potential, the formation of capillary-like tubes, the transendothelial electric resistance (TEER), and the expression of tight junction proteins. RESULTS: HPDL-ECs and HG-ECs with characteristic cobblestone monolayer morphology were obtained, as determined by light microscopy at confluence. Furthermore, the HPDL-ECs and HG-ECs expressed the EC markers platelet endothelial cell adhesion molecule-1 (also known as CD31), von Willebrand factor, and Ulex europaeus agglutinin 1, and the cells stained strongly positive for CD31 and CD309. In addition, the HPDL-ECs and HG-ECs were observed to form capillary-like tubes, and they demonstrated uptake of acetylated low-density lipoprotein. Functional analyses of the HPDL-ECs and HG-ECs showed that, compared to the control cells, tube formation persisted for only a brief period of time, and TEER was substantially reduced at confluence. Furthermore, the cells exhibited delocalization of zonula occludens-1 and occludin at cell-cell contact sites. CONCLUSIONS: The present results provide new evidence that HPDL-ECs and HG-ECs have characteristics of fenestrated capillaries. Therefore, capillaries in human periodontal tissues have functional characteristics of fenestrated capillaries, which might be related to the onset and the progression of systemic diseases and inflammation.

Evaluation of two protocols for low-level laser application in patients submitted to orthodontic treatment.

INTRODUCTION: Different low-level laser (LLL) irradiation protocols have been tested to accelerate orthodontic tooth movement (OTM). Nevertheless, divergent results have been obtained. It was suggested that the stimulatory action of low level laser irradiation occurs during the proliferation and differentiation stages of bone cellular precursors, but not during later stages. OBJECTIVE: The purpose of this study was to determine the effect of two protocols of LLL irradiation on experimental tooth movement: One with daily irradiations and another with irradiations during the early stages. METHODS: Thirty-six rats were divided into control groups (CG1, CG2, CG3) and irradiated groups (IrG1, IrG2, IrG3) according to the presence of: experimental tooth movement, laser irradiation, type of laser irradiation protocol and date of euthanasia (3th or 8th day of experiment). At the end of experimental periods, a quantitative evaluation of the amount of OTM was made and the reactions of the periodontium were analyzed by describing cellular and tissue reactions and by counting blood vessels. RESULTS: The amount of OTM revealed no significant differences between groups in the same experimental period (p < 0.05). Qualitative analysis revealed the strongest resorption activity in irradiated groups after seven days, especially when using the daily irradiation protocol. There was a higher number of blood vessels in irradiated animals than in animals without orthodontic devices and without laser irradiation (p < 0.05). CONCLUSION: Moreover, angiogenesis was verified in some of the irradiated groups. The irradiation protocols tested were not able to accelerate OTM and root resorption was observed while they were applied.

In vivo identification of periodontal progenitor cells.

The periodontal ligament contains progenitor cells; however, their identity and differentiation potential in vivo remain poorly characterized. Previous results have suggested that periodontal tissue progenitors reside in perivascular areas. Therefore, we utilized a lineage-tracing approach to identify and track periodontal progenitor cells from the perivascular region in vivo. We used an alpha-smooth muscle actin (αSMA) promoter-driven and tamoxifen-inducible Cre system (αSMACreERT2) that, in combination with a reporter mouse line (Ai9), permanently labels a cell population, termed 'SMA9'. To trace the differentiation of SMA9-labeled cells into osteoblasts/cementoblasts, we utilized a Col2.3GFP transgene, while expression of Scleraxis-GFP was used to follow differentiation into periodontal ligament fibroblasts during normal tissue formation and remodeling following injury. In uninjured three-week-old SMA9 mice, tamoxifen labeled a small population of cells in the periodontal ligament that expanded over time, particularly in the apical region of the root. By 17 days and 7 weeks after labeling, some SMA9-labeled cells expressed markers indicating differentiation into mature lineages, including cementocytes. Following injury, SMA9 cells expanded, and differentiated into cementoblasts, osteoblasts, and periodontal ligament fibroblasts. SMA9-labeled cells represent a source of progenitors that can give rise to mature osteoblasts, cementoblasts, and fibroblasts within the periodontium.

Evaluation of two protocols for low-level laser application in patients submitted to orthodontic treatment

INTRODUCTION: Different low-level laser (LLL) irradiation protocols have been tested to accelerate orthodontic tooth movement (OTM). Nevertheless, divergent results have been obtained. It was suggested that the stimulatory action of low level laser irradiation occurs during the proliferation and differentiation stages of bone cellular precursors, but not during later stages. OBJECTIVE: The purpose of this study was to determine the effect of two protocols of LLL irradiation on experimental tooth movement: One with daily irradiations and another with irradiations during the early stages. METHODS: Thirty-six rats were divided into control groups (CG1, CG2, CG3) and irradiated groups (IrG1, IrG2, IrG3) according to the presence of: experimental tooth movement, laser irradiation, type of laser irradiation protocol and date of euthanasia (3th or 8th day of experiment). At the end of experimental periods, a quantitative evaluation of the amount of OTM was made and the reactions of the periodontium were analyzed by describing cellular and tissue reactions and by counting blood vessels. RESULTS: The amount of OTM revealed no significant differences between groups in the same experimental period (p < 0.05). Qualitative analysis revealed the strongest resorption activity in irradiated groups after seven days, especially when using the daily irradiation protocol. There was a higher number of blood vessels in irradiated animals than in animals without orthodontic devices and without laser irradiation (p < 0.05). CONCLUSION: Moreover, angiogenesis was verified in some of the irradiated groups. The irradiation protocols tested were not able to accelerate OTM and root resorption was observed while they were applied.

INTRODUÇÃO: diferentes protocolos de irradiação por laser de baixa potência (LBP) têm sido testados para potencializar o movimento ortodôntico; entretanto, há resultados divergentes. Foi sugerido que seu efeito bioestimulador ocorre nas fases de proliferação e diferenciação celular, não agindo em estágios tardios. OBJETIVO: avaliar o efeito de dois protocolos de irradiação do LBP na movimentação ortodôntica: um com irradiações diárias e outro em que irradiações foram realizadas apenas nos períodos iniciais. MÉTODOS: trinta e seis ratos Wistar foram divididos em grupos controles (GC1, GC2 e GC3) e irradiados (GIr1, GIr2 e GIr3), de acordo com a presença de dispositivo ortodôntico, a presença de irradiação, o tipo de protocolo de irradiação e a data de eutanásia (3º ou 8º dia de experimento). Ao final dos períodos experimentais, foram realizadas mensurações da movimentação dentária, análise qualitativa das reações celulares e teciduais do periodonto e contagem de vasos sanguíneos no ligamento periodontal. RESULTADOS: a quantidade de movimentação não diferiu entre os grupos num mesmo tempo experimental (p < 0,05). A análise qualitativa revelou maior atividade absortiva nos grupos irradiados ao final de 7 dias, especialmente quando as irradiações foram diárias. Nos grupos irradiados diariamente, a contagem de vasos foi aumentada em relação aos animais isentos de dispositivo ortodôntico e de aplicações de LBP (p < 0,05). CONCLUSÃO: apesar de verificada angiogênese em certos grupos irradiados, os protocolos de irradiação testados não foram capazes de acelerar a movimentação dentária, e foi possível verificarem-se absorções radiculares.

Comparative studies on microvascular endothelial cells isolated from periodontal tissue.

BACKGROUND: Most available periodontal studies regarding the endothelial cell (EC) were investigated by using human umbilical vein endothelial cells (HUVECs); however, ECs can display remarkable heterogeneity in vascular beds of different origins. The aim of the present study, therefore, is to characterize microvascular ECs isolated from periodontal tissue and investigate their growth and gene expression compared to HUVECs (macrovascular). METHODS: Periodontal ligament ECs (PDL-ECs) and gingiva ECs (G-ECs) were isolated by coupling to monoclonal anti-CD31 antibody magnetic beads. Both PDL-ECs and G-ECs were characterized to definitively demonstrate that the culture represented pure ECs. Their growth was determined by resazurin reduction assay. Interleukin (IL)-8, intercellular adhesion molecule 1 (ICAM-1), and E-selectin gene expression were determined by real-time quantitative reverse-transcription polymerase chain reaction after treatment with Escherichia coli lipopolysaccharide (LPS). RESULTS: PDL-ECs and G-ECs revealed specific EC characteristics but formed tube-like structures and had slower growth rates than HUVECs. After E. coli LPS treatment, PDL-ECs and G-ECs showed similar dose-dependently increased levels of IL-8, ICAM-1, and E-selectin mRNA expression; however, their expressions were in contrast to HUVECs. PDL-ECs and G-ECs showed obviously increased ICAM-1 mRNA expression, whereas HUVECs showed markedly increased E-selectin mRNA expression after treatment with 0.1 µg/mL E. coli LPS. CONCLUSIONS: ECs isolated from periodontal tissue show different growth and gene expression from those of HUVECs. Thus, these microvascular ECs appear to be a more valuable in vitro model system than HUVECs (macrovascular) to further study pathogenesis and angiogenesis of periodontal disease.

Occlusal hypofunction causes periodontal atrophy and VEGF/VEGFR inhibition in tooth movement.

OBJECTIVE: To examine changes in microvasculature and the expression of vascular endothelial growth factor A (VEGF-A) and VEGF receptor 2 (VEGFR-2) in rat hypofunctional periodontal ligament (PDL) during experimental tooth movement. MATERIALS AND METHODS: Twelve-week-old male Sprague-Dawley rats were divided into normal occlusion and occlusal hypofunction groups. After a 2-week bite-raising period, rat first molar was moved mesially using a 10-gf titanium-nickel alloy closed coil spring in both groups. On days 0, 1, 2, 3, and 7 after tooth movement, histologic changes were examined by micro-computed tomography and immunohistochemistry using CD31, VEGF-A, VEGFR-2, and the terminal deoxynucleotidyl transferase mediated dUTP nick end labeling (TUNEL) method. RESULTS: Hypofunctional molars inclined more than normal molars and did not move notably after day 1 of tooth movement. Blood vessels increased on the tension side of the PDL in normal teeth. Immunoreactivities for VEGF-A and VEGFR-2 in normal teeth were greater than those in hypofunctional teeth during tooth movement. Compressive force rapidly caused apoptosis of the PDL and vascular endothelial cells in hypofunctional teeth, but not in normal teeth. CONCLUSIONS: Occlusal hypofunction induces vascular constriction through a decrease in the expression of VEGF-A and VEGFR-2, and apoptosis of the PDL and vascular cells occurs during tooth movement.