Inhibition of osteosarcoma metastasis in vivo by targeted downregulation of MMP1 and MMP9.

Osteosarcoma (OS) mortality stems from lung metastases. Matrix metalloproteinases (MMPs) facilitate metastatic dissemination by degrading extracellular matrix components. Herein we studied the impact of targeted MMP downregulation on OS metastasis. Differential gene expression analysis of human OS cell lines revealed high MMP9 expression in the majority of OS cell lines. Furthermore, 143B, a metastatic OS cell line, exhibited increased MMP1 and MMP9 mRNA levels. Gene set enrichment analysis on metastatic and non-metastatic OS patient specimens indicated epithelial-mesenchymal transition as the most enriched gene set, with MMP9 displaying strong association to genes in this network. Using the same dataset, Kaplan-Meier analysis revealed a correlation between MMP1 expression and dismal patient survival. Hence, we undertook targeted suppression of MMP1 and MMP9 gene expression in OS cell lines. The ability of OS cells to migrate and form colonies was markedly reduced upon MMP1 and MMP9 downregulation, whereas their cell proliferation capacity remained intact. MMP9 downregulation decreased tumor growth and lung metastases area in an orthotopic mouse OS model. Consistently, human OS lung metastasis specimens displayed marked MMP9 protein expression. Our findings highlight the role of MMP1 and MMP9 in OS metastasis, warranting further exploration of simultaneous inhibition of MMPs for future OS therapeutics.

Survival rate and aesthetic outcomes of two-piece zirconia dental implants: A one-year single clinical trial of partially edentulous patients.

INTRODUCTION: Titanium dental implants, traditionally used for tooth replacement, face certain biological and aesthetic limitations. Recently, zirconia has become a notable alternative, valued for its aesthetics and biocompatibility. This study evaluated the efficacy of two-piece zirconia dental implants, particularly their impact on inflammatory cytokines and their survival rate over one year. METHODS: This study was a single-center, prospective trial and included adults aged 18 and above. 2021-2022, nine two-piece tissue-level zirconia implants were placed in eight patients. Following a three-month osseointegration phase, crowns were cemented. Over a year, we assessed Plaque and Gingival indices, Pocket Depth, and tissue color and texture. Peri-apical radiographs measured bone levels, and IL-1ß in peri-implant crevicular fluid (PICF) was quantified using ELISA. RESULTS: Eight subjects (ages 31-63) participated. One implant failed after six months, resulting in a one-year survival rate of 88.8%. Plaque and Gingival indices rose, but peri-implant soft tissue remained stable in color and texture. At 12 months, average bone loss was minimal and insignificant compared to the baseline. IL-1ß levels were similar to those at contralateral teeth, with no correlation between IL-1ß, Pocket Depth, and Bleeding on Probing. CONCLUSION: Two-piece zirconia implants emerged as a viable tooth replacement option, with an 88.8% one-year survival rate. They maintained stable soft tissue and bone levels, indicating their potential as effective dental restoratives.

Tissue changes around dental implants installed in alveolar ridge preservation sites: A 1-year follow-up randomized controlled clinical trial.

OBJECTIVE: This study aimed to assess radiographic marginal bone changes 22 months post extraction, which is 1 year after implant loading in alveolar ridge preservation (ARP) sites grafted with a combination of collagen-embedded xenogenic bone substitute (DBBM-C) and collagen matrix (CMX), comparing them with implants placed in naturally healed sites. METHODS: This randomized controlled clinical trial was conducted over 22 months. Patients who needed a single tooth extraction and subsequent implant placement in nonmolar areas were enrolled. The test group received deproteinized bovine bone mineral with 10% collagen covered by a procaine collagen membrane, while the control group allowed spontaneous healing. Radiographic bone level changes were documented using periapical radiographs at implant placement and follow-up visits (6, 10, and 22 months postextraction). Early implant soft tissue exposure, clinical parameters, and patient-reported outcomes were recorded. RESULTS: Twenty-two out of 28 participants completed a 22-month follow-up, 9 in the test group and 13 in the control group. At 10-month postextraction follow-up, the mean MBL was 1.01 ± 1.04 mm in the treatment group and 0.81 ± 0.93 mm in the control group (p = 0.804). At 22 months, the mean MBL was 2.09 ± 1.03 mm in the treatment group and 1.58 ± 0.73 mm in the control group (p = 0.339). No statistically significant differences in probing depth (PD) and bleeding on probing (BOP) were found at the 22 -month follow-up as well. Soft tissue mean recession was observed in the control group (0.36 ± 0.84 mm), while no recession was found in the test group (p = 0.2). Early implant soft tissue exposure occurred in 33% of test group participants, while none was observed in the control group (p = 0.047). CONCLUSION: One year after implant loading, no significant differences in marginal bone resorption were found between implants placed in ARP-treated and naturally healed sites. However, ARP-treated sites exhibited early implant soft-tissue exposure, suggesting a possible impairment in soft tissue healing.

A novel nonsurgical therapy for peri-implantitis using focused pulsed electromagnetic field: A pilot randomized double-blind controlled clinical trial.

Pulsed electromagnetic field (PEMF) therapy modulates the immune response and is successfully used in orthopedics to treat osteoarthritis and improve bone regeneration. This may suggest that this treatment may consequently reduce peri-implant soft tissue inflammation and marginal bone loss. To compare clinical, radiographic, and immunological results following nonsurgical treatment for peri-implantitis with or without PEMF therapy. Patients with peri-implantitis were included: pocket probing depth (PPD) between 6 and 8 mm with bleeding on probing (BOP); crestal bone loss between 3 and 5 mm. A novel healing abutment that contained active (test) or inactive (control) PEMF was connected. PEMF was administered via the abutment at exposure ratio of 1/500-1/5000, intensity: 0.05-0.5 mT, frequency: 10-50 kHz for 30 days. Nonsurgical mechanical implant surface debridement was performed. Patients were examined at baseline, 1 and 3 months. Clinical assessment included: plaque index, BOP, PPD, recession, and bone crest level which was radiography measured. Samples of peri-implant crevicular fluid were taken to analyze interleukin-1ß (IL-1ß). Twenty-three patients (34 implants; 19 control, 15 test) were included. At the follow-up, mean crestal bone loss was lower in the test group at 1 and 3 months (2.48 mm vs. 3.73 mm, p < 0.05 and 2.39 vs. 3.37, p < 0.01). IL-1ß levels were also lower in the test group at 2 weeks (72.86 pg/mL vs. 111.7, p < 0.05). Within all the limitation of this preliminary study, the test group improved clinical parameters after a short-term period compared to the control group.

Revealing leukocyte populations in human peri-implantitis and periodontitis using flow cytometry.

OBJECTIVE: To identify, quantify, and characterize leukocyte populations in PI and periodontitis using flow cytometry. METHODS: Fresh biopsies from human PI and periodontitis lesions were processed to a single-cell suspension. The immune cell types were identified using flow cytometry. RESULTS: Twenty-one biopsies were obtained and analyzed corresponding to fourteen PI and seven periodontitis samples. Participants' average age was 63.95 ± 14.77 years without a significant difference between PI and periodontitis patients, the female/male ratio was 8/12, and mean PD was 8.5 ± 2.17. High similarity was found between periodontitis and PI in the main immune cell types. Out of the leukocytes, the PMN proportion was 40% in PI and 33% in periodontitis. T-cells 22% in PI and 18% in periodontitis. Similar proportions of B-cells 10% and macrophages 6% were found in PI and periodontitis. Dendritic and NK cells were found in low proportions (~ 1%) in PI and periodontitis. T-cell sub-analysis showed that CD4-positive were more prevalent than CD8-positive in both diseases (CD4/CD8 ratio of 1.2). CONCLUSION: With the use of flow cytometry analysis, the leukocyte populations in human peri-implantitis and periodontitis were classified. In PI and periodontitis, we identified similar proportions of specific (CD4/CD8) and innate (dendritic and NK) immune cells. These results corroborate previous histological studies. CLINICAL RELEVANCE: Flow cytometry analysis can be used to identify and quantify immune cells in PI and periodontitis, including sub-classification of T cells (CD4/8) as well as detection of cells that require multiple markers for identification (such as dendritic cells).

The influence of radio frequency-based toothbrush on the accumulation of calculus and periodontal health: A randomized double-blind controlled prospective study.

OBJECTIVES: The use of a toothbrush with radio frequency (RF) has shown to be of benefit regarding the reduction of plaque, calculus, and dental staining and improving teeth shade compared to conventional powered and manual toothbrushes. AIM: To evaluate the efficacy of the RF toothbrush in the reduction of calculus accumulation and its effect on periodontal parameters as well as subject satisfaction as compared to an identical sham-tooth brush. MATERIALS AND METHODS: Patients who are under a strict maintenance program were included. Patients were allocated to test (RF toothbrush) or control (sham) randomly and were examined at baseline, one and three months. Clinical photos were taken and a consequential calculus assessment via ImageJ software. Clinical assessment included the following: plaque index (PI), bleeding on probing (BOP), probing pocket depth (PPD), and recession (REC). Patient satisfaction was assessed via a questionnaire. RESULTS: Fifty-eight patients (29 control, 29 test) were included. At baseline mean PPD, BOP, PI, REC, and calculus accumulation were similar between the groups. Mean buccal calculus was lower in the test group at one month 4.0% versus 6.7%, p < .05. Calculus accumulation within the groups was lower in the test group at 1 and 3 months when compared to baseline at the buccal aspect (2.8% vs. 8.9%, p < .05% and 3.8% vs. 8.9%, p < .05) and lingual aspect (6.7% vs. 16.5%, p < .05% and 8.9% vs. 16.5%, p < .05). No statistically significant results were found regarding periodontal parameters PPD, BOP, PI, and REC. No difference was found between groups regarding patient satisfaction. CONCLUSION: RF seems to have an additive effect on preventing calculus accumulation on the buccal aspect of anterior mandibular teeth at 1 month. Nevertheless, at 3 months, no difference between the toothbrushes is seen regarding calculus formation and maintaining periodontal health (ClinicalTrials.gov, Identifier NCT04640857).

Endothelial Progenitor Cells Promote Osteosarcoma Progression and Invasiveness via AKT/PI3K Signaling.

BACKGROUND: Osteosarcoma (OS) mortality is attributed to lung metastases. Endothelial progenitor cells (EPCs) mediate the angiogenic switch in several cancers. The spatial proximity between EPCs and OS in the bone led to the hypothesis that EPCs-osteosarcoma interactions may possibly promote OS progression and aggressiveness. METHODS: A PI3K inhibitor, Bevacizumab (an anti-VEGF-A antibody), and an anti-FGF2 antibody were added to the EPCs' conditioned medium (EPC-CM), and their impacts on OS cell (U2-OS and 143B) proliferation, migration, invasion, MMP9 expression, and AKT phosphorylation were determined. The autocrine role of VEGF-A was assessed using Bevacizumab treatment and VEGF-A silencing in OS cells. Toward this end, an orthotopic mouse OS model was established. Mouse and human tumors were immunolabeled with antibodies to the abovementioned factors. RESULTS: EPC-CM enhanced osteosarcoma MMP9 expression, invasiveness, and migration via the PI3K/AKT pathway. The addition of Bevacizumab and an anti-FGF2 antibody to the EPC-CM diminished OS cell migration. The autocrine role of VEGF-A was assessed using Bevacizumab and VEGF-A silencing in OS cells, resulting in decreased AKT phosphorylation and, consequently, diminished invasiveness and migration. Consistently, OS xenografts in mice displayed high VEGF-A and FGF2 levels. Remarkably, lung metastasis specimens derived from OS patients exhibited marked immunolabeling of CD31, VEGF-A, and FGF2. Conclusions: EPCs promote OS progression not only by physically incorporating into blood vessels, but also by secreting cytokines, which act via paracrine signaling. EPCs induced in vitro MMP9 overexpression, invasion, and migration. Additional animal studies are warranted to further expand these results. These findings may pave the way toward the development of novel EPCs-targeted therapeutics aimed at blocking OS metastasis.

Digital photometric analysis of gingival response to periodontal treatment.

OBJECTIVES: The color is a major factor in determining inflammation status in most gingival indices. Current indices have limitations mainly due to subjective nature. Digital color analysis can provide objective and accurate measurements. Thus, the present study aimed to assess by digital tool the gingival color in the different stages of an active periodontal treatment. METHODS: Forty patients (19 males and 21 females) diagnosed with periodontitis (stage III/ IV, grade C) and treated surgically were included in the study. Clinical data (probing depth, bleeding on probing, clinical attachment level, gingival index, and gingival recession) and photographs by digital single-lens-reflex (DSLR) camera were recorded before initial periodontal treatment, which included scaling and root surface debridement (T0); the same parameters were then re-evaluated 6-8 weeks (T1) and 3 months after periodontal surgery (regenerative/resective) (T2). Differences between clinical parameters were calculated. The color space defined by the International Commission on Illumination (CIELab) was used to analyze gingival color. RESULTS: In 56 periodontal surgical sites, 168 photographs were taken. The a*-value of the CIELab color system (higher a*- value translate to a stronger red color) was significantly reduced between T0 to T1 and further decreased at T2 (32.01, 29.28, and 27.45 respectively). Significant improvement in clinical parameters were found between T0 to T1 and T1 to T2. Sub-analysis of two distinct surgical interventions revealed that only regenerative procedure improved the a*-value, which was significantly correlated with pocket depth reduction. CONCLUSIONS: Photometric analysis can be used to assess gingival color change during periodontal treatment of patients with periodontitis. CLINICAL SIGNIFICANCE: Gingival inflammation is a major factor in periodontal assessment; nevertheless, all current gingival inflammation indices are partially subjective and only semi-quantitative. The digital photometric analysis may allow for accurate and objective gingival color assessment during periodontal treatment.

Socket Preservation Using Xenograft Does Not Impair Implant Primary Stability in Sheep: Clinical, Histological, and Histomorphometric Study.

Implant primary stability, which depends mainly on the amount and quality of bone, is important for implant survival. Socket preservation aims to reduce bone volumetric changes after tooth extraction. This animal study aims to examine whether preserving a ridge by using xenograft impairs the primary stability of the implant. Eighteen artificial bone defects were prepared in 4 sheep (5- and 8-mm length). Defects were randomly grafted with xenografts: Bio-Oss (BO), Bio-Active bone (BB), or left for natural healing (control). After 8 weeks, bone biopsy was harvested and dental implants installed. During installation, peak insertion torque (IT) was measured by hand ratchet, and primary stability by the Osstell method. Histomorphometric analysis showed a higher percentage of new bone formation in the naturally healed defects compared to sites with xenograft (control: 68.66 ± 4.5%, BB: 48.75 ± 4.34%, BO: 50.33 ± 4.0%). Connective tissue portion was higher in the BO and BB groups compared to control (44.25 ± 2.98%, 41 ± 6%, and 31.33 ± 4.5%, P < .05, respectively). Residual grafting material was similar in BO and BB (7 ± 2.44%, 8.66 ± 2.1%, respectively). Mean IT and implant stability quotient (ISQ) values were not statistically different among the groups. A positive correlation was found between IT and ISQ (r = 0.65, P = 0). In conclusion, previously grafted defects with xenograft did not influence primary stability and implant insertion torque in delayed implant placement. These results may be attributed to a relatively high bone fill of the defect (∼50%) 2 months after grafting.

A nonsurgical treatment of peri-implantitis using mechanic, antiseptic and anti-inflammatory treatment: 1 year follow-up.

AIMS: The study's aim was to assess the clinical outcome 6 and 12 months after a nonsurgical treatment of peri-implantitis per se or in conjunction with a combination of local antiseptic and anti-inflammatory treatment. MATERIALS AND METHODS: Included were 69 patients with periodontitis, with 106 implants, diagnosed with peri-implantitis. Peri-implantitis was defined as radiographic bone loss ≥3 mm, probing depth (PD) ≥ 6 mm, with bleeding on probing. Group M peri-implantitis was treated with ultrasonic debridement and soft tissue curettage. Group P had additional implant surface treatment with rotatory hand piece composed of chitosan bristle, soft tissue curettage combined with application of 0.95% hypochlorite and 1 mg minocycline HCl. RESULTS: After 6 months, both groups demonstrated significant reduction of mean plaque index, PD, and clinical attachment level (0.71 ± 0.57, 0.81 ± 0.55; 4.77 ± 0.73 mm, 4.42 ± 0.5 mm; 5.03 ± 0.86 mm, 5.13 ± 0.73 mm; respectively) and bleeding on probing. After 6 and 12 months, group P showed significantly better PD results compared to group M. The bleeding was significantly less in group P after 12 months (15.3% ± 6.2, 25.1% ± 8.2, respectively). CONCLUSIONS: Adjunctive treatment with local antiseptic and anti-inflammatories during mechanical phase was positively associated with inflammation reduction and connective tissue reattachment.

Indications for root canal treatment in asymptomatic teeth with various radiographic findings: guidelines for general dental practice.

OBJECTIVES: The purpose of this article was to propose guidelines for dental practitioners facing the dilemma whether to treat, follow up, or disregard random asymptomatic radiographic findings. DATA SOURCES: Searches performed in MEDLINE, Cochrane, and EMBASE databases were followed by a manual search. Related data incorporated by experts included recommendations on asymptomatic root canal treatment, quality of the root canal, and restoration relative to outcome. Evidence tables were developed following quality and inclusion criteria assessment. RESULTS: The initial search retrieved 2,796 MEDLINE, 542 EMBASE, and 152 Cochrane articles, for a total of 3,490 potential articles. After duplicates were removed, 2,946 articles remained. Articles not related to the topic and not meeting eligibility criteria were excluded, resulting in 44 studies included in this scoping review. CONCLUSIONS: The proposed guidelines provide easy access to existing information in endodontics. The findings are common and possess clinical and medico-legal importance.

Biocompatibility and osteoconductivity of PLCL coated and noncoated xenografts: An in vitro and preclinical trial.

BACKGROUND: Cells, scaffolds, and growth factors are the key components in bone tissue engineering. Scaffold composition, topography, and architecture influence the amount of regenerated bone in the implantation site. The aims of the study were to compare viability and proliferation of mesenchymal stem cells (MSCs) seeded onto two commercial xenografts: Bio-Oss (BO) and bioactive bone bovine (BB). Next, these materials were compared for histomorphometric bone formation in a socket preservation model in rats. MATERIALS AND METHODS: MSCs were seeded onto monolayers of BO or BB granules. Cell viability and proliferation were evaluated after incubation of 0, 2, 20, and 48 h. A total of 24 Sprague Dawley rats underwent unilateral extraction of maxillary molars. Rats were randomly divided into three groups: natural healing (nongrafted socket) or socket preservation with either BO or BB. Rats were sacrificed after 8 weeks, and histomorphometric analysis was done to evaluate bone formation and residual scaffold at the extraction site. RESULTS: Differences in the metabolic activity of MSCs that were seeded onto BO or BB was observed at 2 h after seeding: the metabolic activity was elevated compared to baseline in the BB (P = .046) and not changed in the BO wells (P = .84). After 20 h, the metabolic activity of MSCs seeded onto BO was decreasing (P = .005), while cell viability was not changed in the BB group (P = .356). Intergroup comparison revealed higher metabolic activity of MSCs seeded on BB after 48 h compared with BO (P = .016). The in vivo results demonstrated differences in socket healing between the groups: percentage of new bone was higher in the BB compared to BO group (39.1 ± 14.3 vs. 23.7 ± 10.8%, respectively, P = .096). Connective tissue portion was higher in the BO group compared with BB (73.7 ± 11.1 vs. 49.6 ± 13.7%, respectively, P = .018). Residual grafting martial was higher in the BB (11.34 ± 4.18 vs. 2.62 ± 1.23%, P = .011). CONCLUSIONS: The results of this study demonstrating higher vitality and proliferation of MSCs seeded onto BB. Furthermore, following ridge preservation, higher percentage of new bone and lower residual scaffold were found in the BB compared with BO. This enhanced regenerative response might be the result of an enhancement of metabolic activity in cells attached to it. Further research will be needed to understand the precise mechanism.

Photometric CIELAB Analysis of the Gingiva: A Novel Approach to Assess Response to Periodontal Therapy.

BACKGROUND: A new photometric method is introduced for assessment of gingival condition and changes after periodontal therapy. METHODS: Clinical data (probing depth [PD], plaque index [PI], gingival index [GI], bleeding on probing [BOP], and clinical attachment level) and photographs from 53 participants (26 males and 27 females) undergoing initial periodontal treatment were used for this analysis. Each maxillary anterior area was photographed before and 3 months after treatment. Digital image analysis was performed around one incisional tooth, and color data were expressed in terms of L*, a*, and b* values in accordance with the CIELAB color space. Non-parametric Mann-Whitney U test was used for within-group comparisons of disease resolution, as well as means and standard deviations of clinical parameters and ΔL, Δa, and Δb values from baseline to re-evaluation. RESULTS: Significant reduction in mean PI, GI, PD, and BOP were found after treatment in all groups (P <0.001). Significant differences (P <0.05) were recorded by diagnosis of chronic periodontitis in the a* coordinate when comparing treatment results with the baseline (P <0.004). CONCLUSION: Results of this study suggest potential benefit in using photometric analysis to assess gingival changes after therapy.

LL-37 opsonizes and inhibits biofilm formation of Aggregatibacter actinomycetemcomitans at subbactericidal concentrations.

Host defense peptides are immediate responders of the innate immunity that express antimicrobial, immunoregulatory, and wound-healing activities. Neutrophils are a major source for oral host defense peptides, and phagocytosis by neutrophils is a major mechanism for bacterial clearance in the gingival tissue. Dysfunction of or reduction in the numbers of neutrophils or deficiency in the LL-37 host defense peptide was each previously linked with proliferation of oral Aggregatibacter actinomycetemcomitans which resulted in an aggressive periodontal disease. Surprisingly, A. actinomycetemcomitans shows resistance to high concentrations of LL-37. In this study, we demonstrated that submicrocidal concentrations of LL-37 inhibit biofilm formation by A. actinomycetemcomitans and act as opsonins and agglutinins that greatly enhance its clearance by neutrophils and macrophages. Improved uptake of A. actinomycetemcomitans by neutrophils was mediated by their opsonization with LL-37. Enhanced phagocytosis and killing of A. actinomycetemcomitans by murine macrophage-like RAW 264.7 cells were dependent on their preagglutination by LL-37. Although A. actinomycetemcomitans is resistant to the bactericidal effect of LL-37, our results offer a rationale for the epidemiological association between LL-37 deficiency and the expansion of oral A. actinomycetemcomitans and indicate a possible therapeutic use of cationic peptides for host defense.