Periodontal tissue regeneration by recombinant human collagen peptide granules applied with β-tricalcium phosphate fine particles

ObjectivesRecombinant human collagen peptide (RCP) is a recombinantly created xeno-free biomaterial enriched in arginine-glycine-aspartic acid sequences with good processability whose use for regenerative medicine applications is under investigation. The biocompatibility and osteogenic ability of RCP granules combined with β-tricalcium phosphate (TCP) submicron particles (β-TCP/RCP) were recently demonstrated. In the present study, β-TCP/RCP was implanted into experimental periodontal tissue defects created in beagles to investigate its regenerative effects.MethodsAn RCP solution was lyophilized, granulated, and thermally cross-linked into particles approximately 1 mm in diameter. β-TCP dispersion (1 wt%; 500 μL) was added to 100 mg of RCP granules to form β-TCP/RCP. A three-walled intrabony defect (5 mm × 3 mm × 4 mm) was created on the mesial side of the mandibular first molar and filled with β-TCP/RCP.ResultsA micro-computed tomography image analysis performed at 8 weeks postoperative showed a significantly greater amount of new bone after β-TCP/RCP grafting (2.2-fold, P < 0.05) than after no grafting. Histological findings showed that the transplanted β-TCP/RCP induced active bone-like tissue formation including tartaric acid-resistant acid phosphatase– and OCN-positive cells as well as bioabsorbability. Ankylosis did not occur, and periostin-positive periodontal ligament-like tissue formation was observed. Histological measurements performed at 8 weeks postoperative revealed that β-TCP/RCP implantation formed 1.7-fold more bone-like tissue and 2.1-fold more periodontal ligament-like tissue than the control condition and significantly suppressed gingival recession and epithelial downgrowth (P < 0.05).Conclusionsβ-TCP/RCP implantation promoted bone-like and periodontal ligament–like tissue formation, suggesting its efficacy as a periodontal tissue regenerative material.     

Fabrication of calcium phosphate composite polymer/SLS-stabilized emulsion-based bioactive gels and their application for dentine tubule occlusion

ObjectivesCalcium phosphate/SLS/P123 composite bioactive gels were prepared to achieve dentine tubule occlusion.MethodsGels containing calcium phosphate particles were prepared in a water-in-oil microemulsion system with a mixture of triblock copolymer pluronic (P123) as a co-surfactant and sodium lauryl sulfate (SLS) as a surfactant in cyclohexane. Subsequently, calcium chloride dihydrate and sodium hydrogen phosphate aqueous solutions were added in a water phase. Finally, slow evaporation of the oil phase at room temperature was performed to produce a hybrid gel. The obtained gels were investigated for their toxicity by the sulforhodamine B (SRB) assay and applied on human dentine specimens to examine their ability to occlude dentine tubules.ResultsThe size and morphology of the calcium phosphate particles embedded in the gel depended on the concentration of P123 and SLS, which were used as a template for mineral precipitation. The prepared calcium phosphate particles (200–500 nm in diameter) with the maximum polymer and surfactant content exhibited spherical shapes. Further, on reducing their content twice and tenfold yields micro-particles with flower-like shapes. These bioactive gels were able to occlude into dentine tubules after 3 days of application with a plugging rate of 79.22% when using the smallest particles. In addition, calcium phosphate nanorods were transformed into dentine tubules with a maximum depth of 6 μm on increasing the amount of gel.ConclusionsThe bioactive gels were effectively used as bioactive fillers to occlude exposed human dentine tubules.     

Development of a method for the identification of receptor activator of nuclear factor-κB+ populations in vivo

ObjectivesOsteoclasts are induced by macrophage colony-stimulating factor-1 (CSF-1) and receptor activator of nuclear factor-κB (RANK) ligand (RANKL). Monocyte/macrophage lineages are thought to be osteoclast precursors; however, such cells have not been fully characterized owing to a lack of tools for their identification. Osteoclast precursors express colony-stimulating factor-1 receptor (CSF-1R) and RANK. However, the capacity of conventional methods using anti-RANK antibodies to detect RANK⁺ cells by flow cytometry is insufficient. Here, we developed a high-sensitivity method for detecting RANK⁺ cells using biotinylated recombinant glutathione S-transferase-RANKL (GST-RANKL-biotin).MethodsWe sorted sub-populations of mouse bone marrow (BM) or peripheral blood (PB) cells using GST-RANKL-biotin, anti-CSF1R, and anti-B220 antibodies and induced osteoclastogenesis in vitro.ResultsThe frequency of the RANK⁺ population in BM detected by GST-RANKL-biotin was significantly higher than that detected by anti-RANK antibodies. Although RANK⁺ cells were detected in both the B220⁺ and B220^? populations, the macrophage lineage was present only in B220^?. Unexpectedly, a significantly higher number of osteoclasts was induced in RANK^?CSF-1R⁺ cells than in RANK⁺CSF-1R⁺ cells contained in the B220^? population. In contrast, the PB-derived B220^?RANK⁺CSF-1R⁺ population contained a significantly higher frequency of osteoclast precursors than the B220^?RANK^?CSF-1R⁺ population.ConclusionsThese results suggest that GST-RANKL-biotin is useful for the detection of RANK⁺ cells and that RANK and CSF-1R may be helpful indicators of osteoclast precursors in PB.     

Biochemical and X-ray micro-computed tomographic analyses of critical size bone defects grafted with autogenous bone and mercerized bacterial cellulose membranes salified with alendronate

ObjectivesThis study aimed to evaluate the repair of critical-sized bone defects grafted with autogenous bone and mercerized bacterial cellulose membranes (BCm) salified with alendronate (ALN).MethodsForty-eight male Wistar rats underwent surgery to create a 5 mm-diameter bone defect in the calvarium. The removed bone was particularized, regrafted into the defect, and covered by a BCm according to the group: control group (CG), simply mercerized BCm; group 1 (G1), negatively charged BCm (BCm-CM^-) salified with ALN; and group 2 (G2), positively charged BCm (BCm-DEAE⁺) salified with ALN. Serum samples were collected preoperatively and before euthanasia to analyze osteoprotegerin (OPG), parathyroid hormone (PTH), sclerostin (SOST), and fibroblast growth factor 23 (FGF23) levels. The animals were euthanized after 15 or 60 d. Calvaria were analyzed using quantitative microtomography (μCT).ResultsThere was an increased level of PTH in the CG compared to the G2 group, at day 60 (p = 0.019). When analyzing the same group over time, G1 presented an increased FGF23 level on days 15 and 60 (p < 0.05). CG presented an increase in PTH (p = 0.037) at day 60. The μCT analysis detected increased trabecular separation on day 15 in G2 compared to G1 (p = 0.040).ConclusionsSalification of ionized BCm with ALN had no direct effect on bone repair; however, BCm-CM^- increased the levels of FGF23 over time. BCm-DEAE⁺ decreased PTH levels compared to mercerized BCm. BCm-CM-salified with ALN-induced superior bone quality, with respect to trabecular separation, compared to BCm-DEAE⁺.     

Regeneration processes of alveolar bone and microvascular changes after the application of platelet-rich fibrin

ObjectivesPlatelet-rich fibrin (PRF) is a promising agent for bone regeneration (BR). Platelets contain several growth factors that promote angiogenesis and BR. In this study, we observed the morphology of alveolar BR.MethodsPRF (Advanced PRF: A-PRF) was prepared by extracting 10 mL of blood from each dog in a collection tube before tooth extraction. The samples were centrifuged at 200 × g for 8 min and incubated for 10 min to allow clotting. The alveolar socket on the dentition's right side was densely filled with PRF. The opposite side, which did not receive PRF, served as the control group. Different methods were used for specimen preparation and observation. Sections stained with hematoxylin and eosin were observed under a light microscope. Bone specimens were observed using stereoscopic microscopy. The resin cast models were examined using a scanning electron microscope. Moreover, bone formation ratio and height were measured.ResultsFourteen days postoperatively, angiogenesis and bone deposition were more advanced in the PRF group than in the control group. Thirty days postoperatively, both groups developed porous bone. In the PRF group, new bone trabeculae (BT) and a network of blood vessels were formed in the bone marrow. Ninety days postoperatively, the resin cast showed a normal bone structure with BT and bone marrow. Thick BT were observed in the PRF group.ConclusionsGrowth factors in PRF stimulate microcirculation and promote angiogenesis and bone deposition. The benefits of PRF include safety and increased bone formation.     

Characterization and evaluation of ascorbic acid-induced cell sheet formation in human periodontal ligament stem cells: An in vitro study

ObjectivesPeriodontal ligament-derived stem cells (PDLSCs) are regarded as a viable option for periodontal regeneration using cell sheet technology. The objective of the present in vitro study was to characterize human PDLSCs based on their phenotypic and biological properties and to evaluate the ascorbic acid (AA or vitamin C)-induced cell sheet by analyzing the molecular markers.MethodsPDLSCs were established from premolars, and their morphology, viability, proliferation, phenotypic marker expression, and ability to differentiate into osteocytes and adipocytes were analyzed. PDLSCs were then induced to form cell sheets using 100 μM AA, and gene expression was examined by real-time polymerase chain reaction.ResultsPDLSCs showed fibroblastic morphology with >95% viability. The cells were highly proliferative and positive for surface antigens CD29, CD73, and CD90 but negative for CD34 and CD45. They were capable of differentiating into osteocytes and adipocytes. Induction with 100 μM AA transformed PDLSCs into two-to three-layered cell sheets. There was no significant upregulation in ALP and RUNX2 expression in the AA-induced cell sheet. However, the expression levels of late osteoblast differentiation marker (bone gamma-carboxy glutamate protein); cementogenic markers (cementum attachment protein and CP23), and genes encoding extracellular matrix (ECM) proteins [collagen type 1 alpha 1 and integrin beta 1) were higher in AA-induced cell sheets by PDLSCs.ConclusionsThe stimulating effect of AA on cell sheet formation by PDLSCs was confirmed by the expression of typical markers involved in osteogenesis/cementogenesis and ECM secretion, which makes this procedure a prospective option for periodontal tissue regeneration applications.     

Effect of the duration of a conditioned stimulus on component recognition in binary taste mixtures in rats

ObjectivesThe aim of this behavioral study was to investigate the duration of a conditioned stimulus (CS-duration) necessary for rats to recognize the components of a binary taste mixture in a conditioned taste aversion (CTA) paradigm as well as the relationship between CS-duration and their spontaneous recovery.MethodsThe experimental rats were categorized under conditioned and control groups and further divided into three groups according to the CS-duration: 10, 30, and 60 s. As the test stimuli, a mixture of 100 mM sucrose (S) + 30 μM quinine hydrochloride (Q) and its components were used.ResultsOn day 1 of the CTA test, the number of licks (NL) for S + Q and S in all conditioned groups was significantly lower than that of the control group presented with CS for 60 s (CON-60), which was the representative control group determined by the initial CTA test. For Q, there was no significant difference between NL of the CTA group presented with CS for 10 s and that of CON-60; however, NL in the other two CTA groups, i.e., CTA-30 and CTA-60, was significantly lower than that of CON-60. When the rats were presented with a shorter CS-duration, they showed spontaneous recovery earlier depending on the CS-duration.ConclusionsThese results suggest that rats can recognize a binary taste mixture and its components using a CS-duration of more than 30 s and that spontaneous recovery from CTA learning depends on the CS- duration.     

Tooth-level predictors of tooth loss and exposed bone after radiation therapy for head and neck cancer

BackgroundThe objective of this study was to identify tooth-level risk factors for use during preradiation dental care management to predict risk of tooth failure (tooth lost or declared hopeless) and exposed bone after radiation therapy (RT) for head and neck cancer (HNC).MethodsThe authors conducted a prospective observational multicenter cohort study of 572 patients receiving RT for HNC. Participants were examined by calibrated examiners before RT and then every 6 months until 2 years after RT. Analyses considered time to tooth failure and chance of exposed bone at a tooth location.ResultsThe following pre-RT characteristics predicted tooth failure within 2 years after RT: hopeless teeth not extracted pre-RT (hazard ratio [HR], 17.1; P < .0001), untreated caries (HR, 5.0; P < .0001), periodontal pocket 6 mm or greater (HR, 3.4; P = .001) or equaling 5 mm (HR, 2.2; P = .006), recession over 2 mm (HR, 2.8; P = .002), furcation score of 2 (HR, 3.3; P = .003), and any mobility (HR, 2.2; P = .008). The following pre-RT characteristics predicted occurrence of exposed bone at a tooth location: hopeless teeth not extracted before RT (risk ratio [RR], 18.7; P = .0002) and pocket depth 6 mm or greater (RR, 5.4; P = .003) or equaling 5 mm (RR, 4.7; P = .016). Participants with exposed bone at the site of a pre-RT dental extraction averaged 19.6 days between extraction and start of RT compared with 26.2 days for participants without exposed bone (P = .21).ConclusionsIndividual teeth with the risk factors identified in this study should be considered for extraction before RT for HNC, with adequate healing time before start of RT.Practical ImplicationsThe findings of this trial will facilitate evidence-based dental management of the care of patients receiving RT for HNC. This clinical trial was registered at Clinicaltrials.gov. The registration number is NCT02057510.     

The effect of accelerated aging on crystalline structures and optical properties of different monolithic zirconia: A qualitative systematic review

ObjectiveThis study aimed to systematically review the literature related to the impact of low temperature degradation (LTD) on the crystalline structures and optical properties of different types of dental monolithic zirconia materials.MethodsThe systemic review was performed based on the PRISMA statement. In vitro studies investigating the effect of accelerated aging in autoclave (2 bar pressure 134°C – ISO standard 13356-2008) on the crystalline structure and/or optical properties of Yttria-partially stabilized zirconia (Y-PSZ) were included. Specific search terms were used for peer-reviewed articles published in PubMed, Ovid MEDLINE, and EMBASE databases.ResultsFrom 286 eligible articles, 51 articles were selected for full-text analysis, 10 failed to meet the inclusion criteria, and 41 articles were included in this review. Autoclave aging (30 min – 300 h) results in an increase in monoclinic phase (m) content up to 80% for tetragonal zirconia and reaching saturation after 35 h of autoclave aging. All included articles reported less than 1% of monoclinic phase for cubic zirconia after autoclave aging. Translucency parameter was reported between 2.34 and 19.7 after autoclave aging (4–100 h). For same aging time, contrast ratio ranged between 0.48 and 0.95.SignificanceAn increase in monoclinic phase was reported for tetragonal zirconia, while cubic zirconia demonstrates resistance to LTD. The optical properties for all zirconia materials investigated seem more compromised with increasing aging time.     

Adipose-derived mesenchymal stem cells promote salivary duct regeneration via a paracrine effect

ObjectivesThe self-regeneration of exocrine tissues, including salivary glands, is limited and their regeneration mechanism has not yet been fully elucidated. Here we identify the role of adipose-derived mesenchymal stem cells (AMSCs) in salivary gland regeneration.MethodsAMSCs expressing mesenchymal stem cell markers were applied to a submandibular gland injury model and the mechanism of salivary gland repair and regeneration was analyzed.ResultsTransplanted green fluorescent protein (GFP)-labeled AMSCs grew tightly together and promoted ductal regeneration in the regenerative nodule, with slight infiltration of nonspecific immune cells. A comprehensive gene analysis through RNA-sequencing revealed increased expression of bone morphogenetic protein (BMP), transforming growth factor (TGF), and Wnt in AMSC-transplanted regenerative nodules. The factors released from AMSCs scavenge hydrogen peroxidase-induced reactive oxygen species (ROS) through Wnt promoter activity in vitro. Furthermore, AMSC-conditioned medium recovered the growth of the hydrogen peroxidase-damaged primordium of the submandibular gland culture ex vivo.ConclusionsThese results suggest that AMSC-released factors scavenge ROS and maintain salivary gland repair and regeneration via paracrine effects. Thus, AMSCs could be a practical and applicable tool for use in salivary gland regeneration.     

Effects of atorvastatin on sevoflurane postconditioning in in vivo rabbit hearts

ObjectivesMyocardial ischemia-reperfusion injury is a phenomenon that promotes myocardial damage when the blood supply returns to the tissue after a period of ischemia. Anesthetic postconditioning involves myocardial protection against myocardial I/R injury. The effects of atorvastatin (ATV) on sevoflurane postconditioning against myocardial ischemia-reperfusion injury have not been thoroughly studied. The present study aimed to investigate if ATV interacts synergistically with sevoflurane postconditioning against myocardial infarction in rabbit hearts in vivo.MethodsTwenty-eight male rabbits underwent 30 min of left anterior descending coronary artery occlusion that was followed by reperfusion for 180 min under ketamine/xylazine (K/X) anesthesia. Rabbits were randomly assigned to four groups that included Group K/X (under K/X anesthesia only), Group POST (sevoflurane exposure at initial reperfusion), Group ATV (ATV 5 mg/kg/day administered before ischemia), and Group ATV + POST (POST intervention with atorvastatin administered once daily for 3 days). At the end of reperfusion, the myocardial infarct size and the area at risk were both measured.ResultsThe mean infarct sizes in the POST, ATV, and ATV + POST groups were significantly smaller compared to those in the K/X group. Furthermore, the mean infarct size in Group ATV + POST was significantly smaller than was that in Group POST and significantly smaller compared to that in Group ATV.ConclusionThe combination of sevoflurane postconditioning and pre-administration of ATV further reduced the myocardial infarction size compared to that observed with sevoflurane postconditioning alone or ATV alone. Our data suggest that sevoflurane postconditioning and ATV may function additively to enhance cardioprotection.     

In-vitro cytocompatibility of self-adhesive dual-curing resin cements on human mesenchymal stem cells (hMSC) and periodontal ligament cells (PDL-hTERT)

ObjectivesSelf-adhesive dual cured resin cements provide easier clinical application than conventional resin cements but release higher amounts of unreacted monomers, potentially affecting their biocompatibility. This study aimed to compare the cytotoxic effects of self-adhesive dual cured resin cements with two conventional resin cements.MethodsSamples of four resin cements, two self-adhesive dual cured cements (group A: RelyX Unicem, group B: SmartCem), and two conventional resin cements (group C: Panavia 2.0, group D: Variolink Esthetic DC) were prepared with a similar dimension under standardized polymerization conditions and stored in water. For each material 18 samples were used and cell cultures of human mesenchymal stem cells (hMSCs) or periodontal ligament cells (PDL-hTERT) were added under appropriate conditions. One experimental group (group E) was left untreated as control. A cell viability WST test, was performed in each experimental group at day 1, 7, 14 and 21. Moreover, microscopic examination of cells was performed using cell viability staining.ResultsViability of both cell types as determined by WST test was significantly impaired at all time periods by the four different cement materials compared to the untreated control. Comparison between the four materials revealed different inhibition of the viability of both, PDL-hTERT and hMSC cells (group C > group B > group A > group D; p < 0.0001).SignificanceAll resin-based cements caused significant impairment of cell viability, reflecting considerable cytotoxicity. Variolink caused significantly smaller changes of viability than the other tested materials.     

Implementation of repairs in dental practice: Randomized behavior simulation trial

BackgroundDespite increasing evidence, dentists have not widely adopted repairs. The authors aimed to develop and test potential interventions targeting dentists’ behavior.MethodsProblem-centered interviews were performed. Emerging themes were linked to the Behavior Change Wheel to develop potential interventions. The efficacy of 2 interventions was then tested in a postally delivered behavioral change simulation trial among German dentists (n = 1,472 per intervention). Dentists’ stated repair behavior regarding 2 case vignettes was assessed. Statistical analysis was performed using McNemar test, Fisher exact test, and a generalized estimating equation model (P < .05).ResultsTwo interventions (guideline, treatment fee item) were developed on the basis of identified barriers. A total of 504 dentists participated in the trial (17.1% response rate). Both interventions significantly changed dentists’ behavior toward repairs of composite and amalgam restorations, respectively (guideline: difference [Δ] = +7.8% and Δ = +17.6%, treatment fee item: Δ = +6.4% and Δ = +31.5%; adjusted P < .001). Dentists were more likely to consider repairs if they already performed repairs frequently (odds ratio [OR], 1.23; 95% CI, 1.14 to 1.34) or sometimes (OR, 1.08; 95% CI, 1.01 to 1.16), if they regarded repairs as highly successful (OR, 1.24; 95% CI, 1.04 to 1.48), if their patients preferred repairs over total replacements (OR, 1.12; 95% CI, 1.03 to 1.23), for partially defective composite restorations (OR, 1.46; 95% CI, 1.39 to 1.53), and after receiving 1 of the 2 behavioral interventions (OR, 1.15; 95% CI, 1.13 to 1.19).ConclusionsSystematically developed interventions targeting dentists’ repair behaviors are likely efficacious to promote repairs.Practical ImplicationsMost partially defective restorations are replaced completely. Effective implementation strategies are required to change dentists’ behavior.This trial was registered at https://www.clinicaltrials.gov. The registration number is NCT03279874 for the qualitative phase and NCT05335616 for the quantitative phase.     

Vitamin C and eggshell membrane facilitate orthodontic tooth movement and induce histological changes in the periodontal tissue

ObjectivesCollagen remodeling of the periodontal tissue is an important mechanism that involves several biologically active substances to accelerate orthodontic tooth movement. It is known that Vitamin C (VC) enhances collagen production and induces tooth movement. Moreover, the eggshell membrane (ESM) is an integral component of various formulations used to promote wound healing. The purpose of our study was to determine the effects of combined treatment with VC and ESM on periodontal tissues during tooth movement.MethodsNine-week-old male osteogenic disorder Shionogi rats were randomized into four groups: control, VC, ESM, and VC + ESM. The control group was given tap water, and the VC, ESM, and VC + ESM groups were orally administered 0.1% VC solution, 1 wt% ESM solution, and a combination of 0.1 wt% VC and 1 wt% ESM solutions, respectively. A force of 25 or 75 g was applied for 10 days to produce orthodontic tooth movement. Distances of tooth movement were measured on days 3, 7, and 10 of treatment. Histological examination of the periodontal ligament was performed to determine the increase in type I and III collagen levels in response to treatment.ResultsDistances of tooth movement were significantly greater in the VC + ESM group than in the control group. The compression area of the alveolar bone showed increased osteoclastic activity and higher levels of bone resorption in the VC + ESM group. Expression levels of type I and III collagen in the tension area of the alveolar bone were higher in the VC + ESM group than in the control group.ConclusionsThis study revealed that the combined administration of VC and ESM accelerated tooth movement by protecting the periodontal tissue during orthodontic treatment. The combined clinical application of VC and ESM could potentially shorten orthodontic treatment time.