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Background A 6‐week Phase I clinical trial was performed to primarily evaluate the safety and secondarily determine the preliminary efficacy of a novel biological solution, ST266, comprised of a mixture of cytokines, growth factors, nucleic acids, and lipids secreted by cultured amnion‐derived multipotent progenitor cells on gingival inflammation. Methods Fifty‐four adults with gingivitis/periodontitis were randomly assigned to 1X ST266 or diluted 0.3X ST266 or saline topically applied on facial/lingual gingiva (20 µL/tooth). Safety was assessed through oral soft/hard tissue exam, adverse events, and routine laboratory tests. Efficacy was assessed by modified gingival index (MGI), bleeding on probing, plaque index, probing depth (PD), and clinical attachment level (CAL). Assessments were performed on day 0, 8, 12, and 42. ST266 and saline applied daily starting at day 0 through day 12 except weekend days. Plasma was analyzed for safety and proinflammatory cytokines, interleukin (IL)‐1β, IL‐6, tumor necrosis factor‐alpha, and interferon gamma. Gingival crevicular fluid (GCF) was analyzed for the same cytokines. Subgingival plaque was primarily analyzed by checkerboard DNA‐DNA hybridization. Comparisons with saline were modeled through a generalized estimating equations method adjusting for baseline. Results No safety concern was found related to ST266. Statistically significant reduction in MGI was noted at day 42 by 1X ST266 compared with saline (P = 0.044). PD and CAL were reduced by both doses of ST266 at day 42 (P

Background We aimed to evaluate the effect of low doses (LD) bone morphogenetic protein‐2 (BMP2) and BMP4 micro‐immunotherapy (MI) in two in vitro models of periodontal wound healing/regeneration. Methods We first evaluated the effect of LD of BMP2 and BMP4 MI on a 2D cell culture using human gingival fibroblasts (hGF) under inflammatory conditions induced by IL1β. Biocompatibility, inflammatory response (Prostaglandin E2 (PGE2) release), collagen deposition and release of extracellular matrix (ECM) organization‐related enzymes (matrix metalloproteinase‐1 (MMP1) and metalloproteinase inhibitor 1 (TIMP1)) were evaluated after short (3 days) and long‐term (24 days) treatment with BMP2 or BMP4 MI. Then, given the results obtained in the 2D cell culture, LD BMP4 MI treatment was evaluated in a 3D cell culture model of human tissue equivalent of gingiva (GTE) under the same inflammatory stimulus, evaluating the biocompatibility, inflammatory response and effect on MMP1 and TIMP1 release. Results LD BMP4 was able to decrease the release of the inflammatory mediator PGE2 and completely re‐establish the impaired collagen metabolism induced by IL1β treatment. In the 3D model, LD BMP4 treatment improved tissue viability compared with the vehicle, with similar levels to 3D tissues without inflammation. No significant effects were observed on PGE2 levels nor MMP1/TIMP1 ratio after LD BMP4 treatment, although a tendency to decrease PGE2 levels was observed after 3 days. Conclusions LD BMP4 MI treatment shows anti‐inflammatory and regenerative properties on hGF, and improved viability of 3D gingiva under inflammatory conditions. LD BMP4 MI treatment could be used on primary prevention or maintenance care of periodontitis.

BACKGROUND Connective tissue grafting has a beneficial effect on the peri-implant mucosa, but the effect of grafting the buccal mucosa on buccal bone thickness (BBT) has not been investigated, although BBT is proposed to be a key factor for the soft-tissue contour. The aim of this trial was to assess the outcome of a connective tissue graft (CTG) in the aesthetic zone of single immediate implants on the change of BBT according to cone beam computed tomography (CBCT) scan analysis. METHODS In a 1-year randomized controlled trial, 60 patients received an immediately placed implant and provisionalization, either combined with CTG (test group) or without CTG (control group). CBCTs were taken pre-operatively (T$_{pre}$ ) and 1 year after definitive restoration (T$_{2}$ ). Any change in BBT was assessed at different implant levels. Additionally, the change in mid-buccal mucosal level (MBML) and approximal marginal bone level were assessed. RESULTS Fifty-five patients were available for statistical analysis (test group, n = 28; control group, n = 27). At T$_{2}$ , the average change in BBT was significantly larger in the test group (-0.84 ± 0.61 mm) than in the control group (-0.46 ± 0.54 mm, P = 0.02). A MBML gain of 0.07 ± 0.85 mm in the test and a MBML loss -0.52 ± 1.16 mm in the control group was observed at T$_{2}$ . Average loss of marginal bone was 0.05 ± 0.33 mm and 0.01 ± 0.38 mm, respectively. CONCLUSIONS The application of CTG in the aesthetic zone of immediately placed and provisionalized implants is accompanied with more loss of BBT, but at the same time better maintains the mid-buccal mucosal level.

Background Although vitamin D3 deficiency is considered as a risk factor for periodontitis, supplementation during periodontal treatment has not been shown to be beneficial to date. Human periodontal ligament cells (hPDLCs) are regulated by vitamin D3 and play a fundamental role in periodontal tissue homeostasis and inflammatory response in periodontitis. The aim of this study is to investigate possible alterations of the vitamin D3 activity in hPDLCs under inflammatory conditions. Methods Cells isolated from six different donors were treated with either 1,25(OH)2 D3 (0 to 10 nM) or 25(OH)D3 (0 to 100 nM) in the presence and absence of ultrapure or standard Porphyromonas gingivalis lipopolysaccharide (PgLPS), Pam3CSK4, or interferon-γ for 48 hours. Additionally, nuclear factor (NF)-κB inhibition was performed with BAY 11-7082. The bioactivity of vitamin D in hPDLCs was assessed based on the gene expression levels of vitamin D receptor (VDR)-regulated genes osteocalcin and osteopontin. Additionally, VDR and CYP27B1 expression levels were measured. Results The vitamin D3 -induced increase of osteocalcin and osteopontin expression was significantly decreased in the presence of standard PgLPS and Pam3CSK4, which was not observed by ultrapure PgLPS. Interferon-y had diverse effects on the response of hPDLCs to vitamin D3 metabolites. NF-kB inhibition abolished the effects of standard PgLPS and Pam3CSK4. Standard PgLPS and Pam3CSK4 increased VDR expression in the presence of vitamin D3 . CYP27B1 expression was not affected by vitamin D3 and inflammatory conditions. Conclusions This study indicates that the transcriptional activity of VDR is diminished under inflammatory conditions, which might mitigate the effectiveness of vitamin D3 supplementation during periodontal treatment.

Background Ursodeoxycholic acid (UDCA) is one of the first‐line therapeutic medications used in treatment of cholestatic liver disease. Considering that periodontitis is epidemiologically linked to liver diseases, the question arises weather UDCA holds anti‐inflammatory properties on periodontal health. Herein, we provide information that support anti‐inflammatory effects of UDCA on three different periodontium‐related cell types. Methods Gingival fibroblasts and the oral human squamous carcinoma cell line HSC‐2 were exposed to interleukin (IL)1β and tumor necrosis factor (TNF)α with and without UDCA. Murine RAW 264.7 macrophages were incubated with sterile‐filtered human saliva also in the presence of UDCA. The expression of inflammatory cytokines was measured by reverse transcription‐polymerase chain reaction. Immunoassay was applied to detect the production of IL6. Immunostaining was performed for the p65 subunit to further support the anti‐inflammatory role of UDCA. Results We report here that UDCA significantly reduced the IL1β and TNFα‐induced expression of IL1, IL6, and IL8 in gingival fibroblasts and the HSC‐2 cell line. In RAW 264.7 macrophages, UDCA attenuated the expression of IL1α, IL1β, and IL6 that was increased by saliva. Immunoassay confirmed the capacity of UDCA to reduce inflammation‐induced production of IL6 in gingival fibroblasts, HSC‐2 and RAW 264.7 cells. Immunostaining revealed the blocking of nuclear translocation of p65 in gingival fibroblasts. Conclusions Taken together, UDCA can attenuate the provoked expression of inflammatory cytokines in oral fibroblasts, oral human squamous carcinoma cells and macrophages in vitro. These data support the hypothesis that patients with cholestatic liver disease might benefit from UDCA with respect to periodontal health.

Background Basic fibroblast growth factor (bFGF) has been applied for periodontal regeneration. However, the application depends on bone defect morphology because bFGF diffuses rapidly from defect sites. In a previous study, collagen‐binding bFGF (CB‐bFGF) has been shown to enhance bone formation by collagen‐anchoring in the orthopedic field. The aim of this study is to demonstrate the efficacy of CB‐bFGF with collagen scaffolds in bone regeneration of horizontal bone defect. Methods Cell proliferation activity and collagen binding activity of CB‐bFGF was confirmed by WST‐8 assay and collagen binding assay, respectively. The retention of CB‐bFGF in the collagen sheet (CS) was measured by fluorescence imaging. The rat horizontal alveolar bone defect model was employed to investigate the efficacy of CB‐bFGF with collagen powder (CP). After 4 and 8 weeks, the regenerative efficacy was evaluated by microcomputed tomography, histological, and immunohistochemical analyses. Results CB‐bFGF had a comparable proliferation activity to bFGF and a collagen binding activity. CB‐bFGF was retained in CS longer than bFGF. At 8 weeks postoperation, bone volume, bone mineral content, and new bone area in CB‐bFGF/CP group were significantly increased compared with those in other groups. Furthermore, epithelial downgrowth was significantly suppressed in CB‐bFGF/CP group. At 4 weeks, the numbers of osteocalcin, proliferating cell nuclear antigen, and osteopontin‐positive cells at the regeneration site in CB‐bFGF/CP group were greater than those in other groups. Conclusions CB‐bFGF/CP effectively promoted bone regeneration of horizontal bone defect possibly by sustained release of bFGF. The potential of CB‐bFGF composite material for improved periodontal regeneration in vertical axis was shown.

BACKGROUND: To evaluate whether buccal bone thickness (BBT), implant diameter, and abutment/crown material influence the accuracy of cone beam computed tomography (CBCT) to determine the buccal bone level at titanium implants.METHODS: Two implant beds (i.e., narrow and standard diameter) were prepared in each of 36 porcine bone blocks. The implant beds were positioned at a variable distance from the buccal bone surface, thus resulting in 3 BBT groups (i.e., > 0.5-1.0; > 1.0-1.5; > 1.5-2.0 mm). In half of the blocks, a buccal bone dehiscence of random extent ("depth") was created and implants were mounted with different abutment/crown material (i.e., titanium abutments with a metal-ceramic crown and zirconia abutments with an all-ceramic zirconia crown). The distance from the implant shoulder to the buccal bone crest was measured on cross-sectional CBCT images and compared to the direct measurements at the bone blocks.RESULTS: While abutment/crown material and implant diameter had no effect on the detection accuracy of the buccal bone level at dental implants in CBCT scans, BBT had a significant effect. Specifically, when BBT was ≤ 1.0 mm, a dehiscence was often diagnosed although not present, i.e., the sensitivity was high (95.8%), but the specificity (12.5%) and the detection accuracy (54.2%) were low. Further, the average measurement error of the distance from the implant shoulder to the buccal bone crest was 1.6 mm.CONCLUSIONS: Based on the present laboratory study, BBT has a major impact on the correct diagnosis of the buccal bone level at dental titanium implants in CBCT images; in cases where the buccal bone is ≤ 1 mm thick, detection of the buccal bone level is largely inaccurate. This article is protected by copyright. All rights reserved.

Background MicroRNAs (miRNAs) are small noncoding RNAs demonstrated as critical post‐transcriptional modulators in dental tissues and bone regeneration, particularly miR‐21‐5p. However, the role of miR‐21‐5p in the healing of alveolar sockets following tooth extraction remains unknown. In this study we evaluated the influence of miR‐21‐5p in the healing of alveolar socket after tooth extraction. Methods Eight miR‐21‐5p knockout mice and eight littermate controls underwent tooth extraction of the upper right incisor. After a healing period of 14 days microCT and histological analyses were performed. Results MicroCT analysis showed that the percentage of bone in the extraction socket was significantly higher in the control group than in the miR‐21 knockout mice; either in the coronal (39.0%, CI 31.8 to 48.0 versus 23.0%, CI 17.8 to 35.2, P = 0.03) or in the middle part of the alveolar socket (56.0%, CI 50.9 to 62.5 versus 43.5% CI 28.6 to 54.6, P = 0.03). These differences were not noted in the apical part of the extraction socket. Histological analysis supported the microCT findings. Newly bone volume per tissue volume (BV/TV) was significantly higher in the control group when compared to miR‐21 knockout mice, 27.4% (CI 20.6 to 32.9) versus 19.0% (CI 14.7 to 21.5, P

Background Platelet‐rich fibrin (PRF) serves as a reservoir of bioactive molecules to support wound healing and bone regeneration. The beneficial action of PRF might involve macrophage polarization from proinflammatory M1 toward pro‐resolving M2 phenotypes. This study aims to evaluate the effect of PRF on macrophage polarization. Methods Murine primary macrophages and RAW 264.7 cells were exposed to saliva and lipopolysaccharides (LPS) with and without PRF lysates obtained by repeated freeze‐thawing or the secretome of PRF membranes, termed PRF conditioned medium. The expression of the M1 marker genes interleukin 1β (IL1β) and interleukin 6 (IL6) along with the M2 markers arginase‐1 and chitinase‐like 3 (Chil3 or YM1) were evaluated by real time polymerase chain reaction. Immunoassay and immunofluorescence staining were performed for IL6 and p65 translocation, a subunit nuclear factor kappa‐light‐chain‐enhancer of activated B cells (NF‐kB), respectively. Results We report here that PRF lysates and PRF conditioned medium, the latter containing the secretome, greatly decreased the proinflammatory response of primary macrophages and RAW 264.7 cells as indicated by the expression of IL1β and IL6. The anti‐inflammatory activity of PRF lysates was further confirmed by IL6 immunoassay. Moreover, PRF lysates suppressed the translocation of p65 from the cytoplasm into the nucleus after incubation with saliva. In support of M2 polarization, PRF lysates and PRF conditioned medium enhanced the expression of arginase‐1 and YM1 in primary macrophages. Conclusion Our results indicate that PRF holds an anti‐inflammatory activity and shifts the macrophage polarization from an M1 toward an M2 phenotype.

Background Platelet‐rich fibrin (PRF) membranes can preserve alveolar ridge dimension after tooth extraction. Thus, it can be presumed that PRF suppresses the catabolic events that are caused by osteoclastic bone resorption. Methods To address this possibility, we investigated the impact of soluble extracts of PRF membranes on in vitro osteoclastogenesis in murine bone marrow cultures. Osteoclastogenesis was induced by exposing murine bone marrow cultures to receptor activator of nuclear factor kappa B ligand (RANKL), macrophage colony‐stimulating factor (M‐CSF) and transforming growth factor‐beta 1 (TGF‐β1) in the presence or absence of PRF. Osteoclastogenesis was evaluated based on histochemical, gene expression, and resorption analysis. Viability was confirmed by formation of formazan crystals, live‐dead staining and caspase‐3 activity assay. Results We report here that in vitro osteoclastogenesis is greatly suppressed by soluble extracts of PRF membranes as indicated by tartrate‐resistant acid phosphatase (TRAP) staining and pit formation. In support of the histochemical observations, soluble extracts of PRF membranes decreased expression levels of the osteoclast marker genes TRAP, Cathepsin K, dendritic cell‐specific transmembrane protein (DCSTAMP), nuclear factor of activated T‐cells (NFATc1), and osteoclast‐associated receptor (OSCAR). PRF membranes, however, cannot reverse the process once osteoclastogenesis has evolved. Conclusion These in vitro findings indicate that PRF membranes can inhibit the formation of osteoclasts from hematopoietic progenitors in bone marrow cultures. Overall, our results imply that the favorable effects of PRF membranes in alveolar ridge preservation may be attributed, at least in part, by the inhibition of osteoclastogenesis.

Background Although periodontitis is associated with disruption of the host‐microbial homeostasis, viruses are currently discussed to influence disease progression. Viral pathogens are recognized by Toll‐like receptor (TLR)‐3, which engages a different signaling pathway than other TLRs. This study aimed to investigate the effect of TLR‐3 agonist polyinosinic:polycytidylic acid (Poly I:C) on the expression of inflammatory markers and bone metabolism proteins by human periodontal ligament stem cells (hPDLSCs) compared with TLR‐2 agonist Pam3CSK4, which mimics the effect of bacterial lipoproteins. To assess potential combined effects of bacterial and viral infections, hPDLSCs response to simultaneous TLR‐2 and TLR‐3 activation was investigated. Methods HPDLSCs were stimulated with Poly I:C (0.0001‐1 µg/mL), Pam3CSK4 (1 µg/mL), and their combinations for 24 hours. Gene expression and protein levels of interleukin (IL)‐6, IL‐8, monocyte chemoattractant protein (MCP)‐1, and osteoprotegerin (OPG) were measured with qPCR and ELISA. Results Production of IL‐6, IL‐8, MCP‐1, and OPG was significantly increased by Poly I:C or Pam3CSK4 to a similar extent. The levels of all inflammatory mediators induced by simultaneous stimulation with Poly I:C and Pam3CSK4 were significantly higher compared with single stimuli as well as to their summed response. Gene expression and protein levels of OPG were enhanced by Poly I:C, but by lesser extent than by Pam3CSK4. OPG levels upon simultaneous stimulation with Pam3CSK4 and Poly I:C were significantly lower compared with Pam3CSK4 stimulation alone. Conclusions Simultaneous TLR‐2 and TLR‐3 activation synergistically triggers IL‐6, IL‐8, and MCP‐1 production, which was not observed for OPG. These findings suggest that TLR‐3 activation by viral infections might promote periodontitis progression.