Contribution of initial lymphatics to oral wound healing after tooth extraction.

Lymphatics are involved in the resolution of inflammation and wound healing, but their role in the oral wound healing process after tooth extraction has never been investigated. We therefore sought to evaluate the healing process following the extraction of maxillary molars in two transgenic mouse models: K14-VEGFR3-Ig mice, which lack initial mucosal lymphatic vessels, and K14-VEGFC mice, which have hyperplastic mucosal lymphatics. Maxillary molars were extracted from both transgenic mouse types and their corresponding wild-type (WT) controls. Mucosal and alveolar bone healing were evaluated. A delayed epithelialization and bone regeneration were observed in K14-VEGFR3-Ig mice compared with their WT littermates. The hampered wound closure was accompanied by decreased levels of epidermal growth factor (EGF) and persistent inflammation, characterized by infiltrates of immune cells and elevated levels of pro-inflammatory markers in the wounds. Hyperplastic mucosal lymphatics did not enhance the healing process after tooth extraction in K14-VEGFC mice. The findings indicate that initial mucosal lymphatics play a major role in the initial phase of the oral wound healing process.

Human gingival epithelial cells stimulate proliferation, migration, and tube formation of lymphatic endothelial cells in vitro.

OBJECTIVE: The aim of this study was to investigate the response of gingival epithelial cells to microbial and inflammatory signals. BACKGROUND: The gingival epithelial barrier provides the first line of defense and supports tissue homeostasis by maintaining the cross-talk between gingival epithelium, oral microbiota, and immune cells. Lymphatic vessels are essential to sustaining this homeostasis. The gingival epithelial cells have been shown to produce prolymphangiogenic factors during physiologic conditions, but their role in response to microbial and inflammatory signals is unknown. METHODS: Immortalized human gingival epithelial cells (HGEC) and human dermal lymphatic microvascular endothelial cells (LEC) were cultured. HGEC were exposed to Porphyromonas gingivalis derived-LPS, human IL-1 beta/IL-1F2 protein, or recombinant human IL-6/IL-6R. Levels of vascular growth factors (VEGF-A, VEGF-C, and VEGF-D) in cell supernatants were determined by ELISA. LEC were grown to confluence, and a scratch was induced in the monolayer. Uncovered area was measured up to 48 h after exposure to conditioned medium (CM) from HGEC. Tube formation assays were performed with LEC cocultured with labelled HGEC or exposed to CM. RESULTS: VEGF-A, VEGF-C, and low levels of VEGF-D were constitutively expressed by HGEC. The expression of VEGF-C and VEGF-D, but not VEGF-A, was upregulated in response to proinflammatory mediators. VEGF-C was upregulated in response to P. gingivalis LPS, but not to Escherichia coli LPS. A scratch migration assay showed that LEC migration was significantly increased by CM from HGEC. Both the CM and coculture with HGEC induced significant tube formation of LEC. CONCLUSIONS: HGEC can regulate production of lymphangiogenic/angiogenic factors during inflammatory insults and can stimulate proliferation, migration, and tube formation of LEC in vitro in a paracrine manner.

Fluid transport from the dental pulp revisited.

In the dental pulp surrounded by rigid dentinal walls, an increase in fluid volume will be followed by a rapid increase in interstitial fluid pressure. To maintain pressure homeostasis, a fluid drainage system is required. The dental pulp and apical periodontal ligament lack lymphatic vessels, and the questions are how the transport can take place inside the pulp and where the lymphatic vessels draining fluid from the apical periodontal ligament are located. The drainage of fluid within the pulp must be governed by a tissue pressure gradient (driving pressure) and the fluid is likely transported in loose connective tissue (gaps) surrounding vessels and nerve fibers. We suggest that aging of the pulp tissue characterized by fibrosis will reduce the draining capacity and make it more vulnerable to circulatory failure. When the fluid leaves the pulp, it will follow the nerve bundles and vessels through the periapical ligament into bone channels, where lymphatic vessels are found. In the mandibular canal, lymphatic vessels are localized and the fluid washout rate from the canal is slow, but chewing may speed it up by increasing the fluid pressure. In acute apical periodontitis, inflammatory mediators and bacterial components can be spread to regional lymph nodes via lymphatic vessels inside the jaw bone.

Interstitial fluid drainage from rat apical area takes place via vessels in the mandibular canal.

We sought to investigate the transport route for protein-rich fluid from the apical area towards the draining lymph nodes. The first mandibular molar root canals in 24 female Wistar rats were instrumented and filled with radioactive-labelled human serum albumin. The rats were sacrificed at different intervals beginning after 10 min (time 0) and continuing up to 72 h. Three jaw segments, gingiva around the first molar, blood samples, submandibular and cervical lymph nodes were collected and analyzed for radioactivity. The starting volume of tracer (control) for all experiments was calculated from measurements at time 0. At time 0, radioactivity was only detected in the jaw segments. Within lymph nodes and serum, the tracer was found after 4 h, with the highest amount recorded in serum up to 24 h. Lymphatics were found within the mandibular canal along blood vessels and nerves and exiting via foramen mandibularis, after immunohistochemical staining in four untreated rats. Our results show tracer distribution from the apical area towards the mandibular canal in a posterior direction. The tracer washout rate was low, and the fluid was mainly absorbed into blood vessels. The lymphatics in the mandibular canal may be more important for immune cell transport than for fluid drainage.

Vascular endothelial growth factors signalling in normal human dental pulp: a study of gene and protein expression.

In the well-vascularized dental pulp vascular endothelial growth factor A (VEGF-A) is expressed. Vascular endothelial growth factor A is a member of the VEGF family, which includes VEGFs-B, -C, and -D. The latter three have not been investigated in the pulp. Vascular endothelial growth factors C and D are the only ligands for vascular endothelial growth factor receptor (VEGFR)-3, which is usually expressed in lymphatic endothelium. They can also activate VEGFR-2, the main angiogenic receptor. We aimed to study VEGFs signalling in human dental pulp at the gene level and to identify the cellular source for protein expression using immunolabelling. All VEGFs (-A, -B, -C, and -D) were expressed in the pulp and may exert both autocrine and paracrine effects in blood vessels and immune cells found to be equipped with VEGFRs-2 and -3. Lymphatic vessel endothelial hyaluronan receptor-positive macrophages, known to be involved in angiogenesis, were found in the pulp, whereas lymphatic vessels were not detected. Twenty-six of 84 VEGF signalling genes, including VEGFR-3, were expressed at a significantly higher level in the pulp than in the control periodontal ligament. In conclusion, the normal human pulp represents a tissue with relatively high VEGF signalling involving both immune responses and vascular activity.

Biomarkers common for inflammatory periodontal disease and depression: A systematic review.

Background: Dysregulated immune response arising in the periphery can induce depressive symptoms through neuroimmune interactions. Inflammatory oral pathology can be a potent inducer of chronic neuroimmune response relevant to depression. We aimed to synthesize available evidence for the association between inflammatory periodontal diseases (IPD) and major depression (MD) in relation to a broad range of biomarkers. Methods: Medline, Embase, PsycInfo, Cochrane Library, Web of Science and Scopus databases were searched from inception until January 27, 2022. Search terms included subject headings and synonyms for inflammatory periodontal disease and depression. Studies that reported data on both depression and inflammatory periodontal disease as categories along with measurement of a biomarker were considered. Two reviewers independently selected the articles for inclusion, extracted data and assessed the quality of each study. The protocol for this study was registered with PROSPERO, CRD42021215524. Results: Twenty-eight studies were included in the final review-eleven cross-sectional studies, seven case-control studies, and six prospective cohort studies conducted in humans; the remaining four were experimental animal studies. Eighteen studies including all animal studies reported a positive association between depression and periodontal disease; one study reported a negative association and another nine studies found no such associations. Twenty studies reported mixed associations between IPD and biomarkers (i.e, salivary, serum, urine or gingival crevicular fluid cortisol, C reactive protein, cytokines, etc.). Biomarkers related to depression were gingival crevicular fluid cortisol, interleukin 6 (IL-6), Il-1ß, immunoglobulin G against Bacterioides forsythus; root canal lipopolysaccharides; blood IL-6, IL-1ß, cortisol, advanced oxidation protein products, nitric oxide metabolites, lipid hydroperoxides and trapping antioxidant parameter; whereas five studies found no associations between depression and a biomarker. Although animal studies showed interaction of immune, inflammatory and neurotrophic biomarkers in the relationship between depression and periodontal disease, human studies showed mixed findings. In most studies, there were risks of bias due to the sample selection and assessment protocol. Study heterogeneity and limited number of comparable studies reporting on shared biomarkers precluded a meta-analysis. Conclusion: Immune-inflammatory contribution to depression was evident in the context of inflammatory periodontal diseases, but whether biomarkers mediate the associations between IPD and MD needs to be tested through methodologically rigorous studies aiming specifically at this hypothesis.

Determination of the performance of various root canal disinfection methods after in situ carriage.

The aim of this study was to compare the antimicrobial performance of the Endox Endodontic System (Lysis Srl, Nova Milanese, Italy), MTAD (Dentsply Tulsa Dental, Tulsa, OK), sodium hypochlorite (NaOCl), and HealOzone (Kavo, Biberach, Germany). Seventy instrumented and initially sterile roots with open accesses and containing a paper point were carried by one volunteer in the oral cavity for 1 week. After removal, samples were taken for microbiological analysis. The root canals were then disinfected with the Endox Endodontic System, MTAD, 3% NaOCl, or HealOzone, and, thereafter, samples were repeated for microbiological analysis. The roots were then sealed and incubated for a further week, after which bacterial growth was again determined. After disinfection, there was a significant decrease in the absolute bacterial count between each disinfection method and the positive control group. There was no statistically significant difference between the NaOCl, MTAD, and HealOzone groups. The Endox device showed the least antibacterial effect with significant differences to MTAD and HealOzone. Bacterial regrowth after 1 week of incubation was detected in all samples of the control group, whereas test groups showed several bacteria-free samples.

VEGFR-2 reduces while combined VEGFR-2 and -3 signaling increases inflammation in apical periodontitis.

BACKGROUND: In apical periodontitis, oral pathogens provoke an inflammatory response in the apical area that induces bone resorptive lesions. In inflammation, angio- and lymphangiogenesis take place. Vascular endothelial growth factors (VEGFs) and their receptors (VEGFRs) are key players in these processes and are expressed in immune cells and endothelial cells in the lesions. OBJECTIVE: We aimed at testing the role of VEGFR-2 and -3 in periapical lesion development and investigated their role in lymphangiogenesis in the draining lymph nodes. DESIGN: We induced lesions by pulp exposure in the lower first molars of C57BL/6 mice. The mice received IgG injections or blocking antibodies against VEGFR-2 (anti-R2), VEGFR-3 (anti-R3), or combined VEGFR-2 and -3, starting on day 0 until day 10 or 21 post-exposure. RESULTS: Lesions developed faster in the anti-R2 and anti-R3 group than in the control and anti-R2/R3 groups. In the anti-R2 group, a strong inflammatory response was found expressed as increased number of neutrophils and osteoclasts. A decreased level of pro-inflammatory cytokines was found in the anti-R2/R3 group. Lymphangiogenesis in the draining lymph nodes was inhibited after blocking of VEGFR-2 and/or -3, while the largest lymph node size was seen after anti-R2 treatment. CONCLUSIONS: We demonstrate an anti-inflammatory effect of VEGFR-2 signaling in periapical lesions which seems to involve neutrophil regulation and is independent of angiogenesis. Combined signaling of VEGFR-2 and -3 has a pro-inflammatory effect. Lymph node lymphangiogenesis is promoted through activation of VEGFR-2 and/or VEGFR-3.

Localization and signaling patterns of vascular endothelial growth factors and receptors in human periapical lesions.

INTRODUCTION: Vascular endothelial growth factors (VEGFs) and their receptors (VEGFRs) are key players in vasculogenesis and are also involved in pathologic conditions with bone destruction. Vasculogenesis is critical for disease progression, and bone resorption is a hallmark of apical periodontitis. However, the localization of VEGFs and VEGFRs and their gene signaling pathways in human apical periodontitis have not been thoroughly investigated. The aim of this study was to localize VEGFs and VEGFRs and analyze their gene expression as well as signaling pathways in human periapical lesions. METHODS: Tissue was collected after endodontic surgery from patients diagnosed with chronic apical periodontitis. Periodontal ligament samples from extracted healthy wisdom teeth was also collected and used as control tissue. In lesion cryosections, VEGFs/VEGFRs were identified by immunohistochemistry/double immunofluorescence by using specific antibodies. A human VEGF signaling polymerase chain reaction array system was used for gene expression analysis comparing lesions with periodontal ligament samples. RESULTS: The histologic evaluation revealed heterogeneous morphology of the periapical lesions with various degrees of inflammatory infiltrates. In the lesions, all investigated factors and receptors were identified in blood vessels and various immune cells. No lymphatic vessels were detected. Gene expression analysis revealed up-regulation of VEGF-A and VEGFR-3, although not significant. Phosphatidylinositol-3-kinases, protein kinase C, mitogen-activated protein kinases, and phospholipases, all known to be involved in VEGF-mediated angiogenic activity, were significantly up-regulated. CONCLUSIONS: The cellular and vascular expressions of VEGFs and VEGFRs in chronic apical periodontitis, along with significant alterations of genes mediating VEGF-induced angiogenic responses, suggest ongoing vascular remodeling in established chronic periapical lesions.

Vascular endothelial growth factors and receptors are up-regulated during development of apical periodontitis.

INTRODUCTION: Apical periodontitis is a common inflammatory disease caused by persistent root canal infection and is characterized by bone resorption. Vascular endothelial growth factors (VEGFs) and their receptors (VEGFRs) have been described in many pathologic and inflammatory conditions, but their involvement in the development of apical periodontitis has not been thoroughly investigated. The aim of this study was to quantify gene expression and localize VEGF-A, VEGF-C, and VEGF-D and VEGFR-2 and VEGFR-3 in a rat model of apical periodontitis. METHODS: Molar pulps were unilaterally exposed to the oral cavity for 10 or 21 days. Jaw sections were used for localization of VEGFs and VEGFRs with immunohistochemistry and identification of cells with double immunofluorescence. Gene expression analysis for VEGF-A, VEGF-C, and VEGFR-3 of periapical tissues was performed with quantitative real-time polymerase chain reaction. RESULTS: All investigated factors and receptors were expressed immunohistochemically in blood vessels at the periodontal ligament of control teeth and were up-regulated during lesion development. In apical lesions, macrophages and neutrophils expressed all studied factors and receptors, with macrophages being an important source of VEGF-C and VEGF-D. Osteoclasts expressed VEGFR-2 and VEGFR-3, and the latter was also identified in fibroblast-like cells in the lesions. VEGF-A and VEGFR-3 gene expression was up-regulated at days 10 and 21 (P < .05). CONCLUSIONS: The current findings indicate that the VEGF family and receptors are involved in vascular remodeling and immune functions during disease development. The presence of VEGFR-2 and VEGFR-3 on osteoclasts indicates that bone resorbing activity is influenced by VEGFs.

Preparation of the coronal and middle third of oval root canals with a rotary or an oscillating system.

OBJECTIVES: To comparatively evaluate the preparation of oval root canals with a rotary or an oscillating system. STUDY DESIGN: The middle and coronal parts of 55 extracted permanent teeth with oval canals were prepared using FlexMaster (FM) rotary NiTi instruments and EndoEze AET (EE) stainless steel oscillating instruments. Pre- and postoperative images of cross-sections were superimposed to identify shifts in the center and to assess the percentage of untreated regions. In addition, the middle segment was investigated by scanning electron microscope (SEM) to determine debris and smear layer removal. RESULTS: The systems did not significantly differ in the shifts of the canal centers in the middle part of the root. Only a few of the preparations yielded an excellent result with no uninstrumented canal wall left. The SEM investigation demonstrated poor results for both systems regarding debris and smear layer removal, but no significant differences could be observed. CONCLUSIONS: Neither FM nor EE was capable of completely preparing oval root canals.