In Vitro Investigation of Gelatin/Polycaprolactone Nanofibers in Modulating Human Gingival Mesenchymal Stromal Cells.

The aesthetic constancy and functional stability of periodontium largely depend on the presence of healthy mucogingival tissue. Soft tissue management is crucial to the success of periodontal surgery. Recently, synthetic substitute materials have been proposed to be used for soft tissue augmentation, but the tissue compatibility of these materials needs to be further investigated. This study aims to assess the in vitro responses of human gingival mesenchymal stromal cells (hG-MSCs) cultured on a Gelatin/Polycaprolactone prototype (GPP) and volume-stable collagen matrix (VSCM). hG-MSCs were cultured onto the GPP, VSCM, or plastic for 3, 7, and 14 days. The proliferation and/or viability were measured by cell counting kit-8 assay and resazurin-based toxicity assay. Cell morphology and adhesion were evaluated by microscopy. The gene expression of collagen type I, alpha1 (COL1A1), α-smooth muscle actin (α-SMA), fibroblast growth factor (FGF-2), vascular endothelial growth factor A (VEGF-A), transforming growth factor beta-1 (TGF-ß1), focal adhesion kinase (FAK), integrin beta-1 (ITG-ß1), and interleukin 8 (IL-8) was investigated by RT-qPCR. The levels of VEGF-A, TGF-ß1, and IL-8 proteins in conditioned media were tested by ELISA. GPP improved both cell proliferation and viability compared to VSCM. The cells grown on GPP exhibited a distinct morphology and attachment performance. COL1A1, α-SMA, VEGF-A, FGF-2, and FAK were positively modulated in hG-MSCs on GPP at different investigation times. GPP increased the gene expression of TGF-ß1 but had no effect on protein production. The level of ITG-ß1 had no significant changes in cells seeded on GPP at 7 days. At 3 days, notable differences in VEGF-A, TGF-ß1, and α-SMA expression levels were observed between cells seeded on GPP and those on VSCM. Meanwhile, GPP showed higher COL1A1 expression compared to VSCM after 14 days, whereas VSCM demonstrated a more significant upregulation in the production of IL-8. Taken together, our data suggest that GPP electrospun nanofibers have great potential as substitutes for soft tissue regeneration in successful periodontal surgery.

Optimized Erbium-Doped Yttrium Aluminum Garnet (Er:YAG) Laser Parameters for the Removal of Dental Ceramic Restorations.

OBJECTIVES: The use of lasers for debonding adhesively luted ceramic restorations is a rather recent oral laser application in dentistry. The removal of all-ceramic restorations in the mouth can often be a troublesome task. A novel method for the debonding of ceramic restorations without damaging the restorations is Er:YAG laser irradiation. The aim of this study was to evaluate the Er:YAG laser for debonding procedures of different dental ceramics and to identify appropriate laser settings. MATERIAL AND METHODS: Lithium disilicate, zirconium-reinforced lithium silicate, feldspatic ceramic, and zirconium dioxide were investigated. Ten ceramic rectangular-shaped specimens with 1 and 2 mm thickness were produced from each material. All specimens were irradiated with four different power settings 1.5; 2.5; 3.5; 4.5 W, pulse duration 50 µs, laser repetition rate 10 Hz, time of irradiation 10 s. The transmitted energy was measured with a powermeter. Additionally the suitability of the Er:YAG laser to remove the adhesively bonded ceramic and the time until loss of retention was evaluated. RESULTS: The transmission rate for 1 and 2 mm platelets was determined for zirconium-reinforced lithium silicate at 54.6%/35.6%, lithium disilicate at 53.2%/35.7%, zirconium dioxide at 40.6%/32.4%, and for the feldspathic ceramic at 19.4%/10.1%. For zirconium-reinforced lithium silicate and zirconium dioxide 2.5 W (250 mJ/10 Hz) was an appropriate energy level for effective debonding. Whereas for lithium disilicate and for feldspathic ceramic, 4.5 W (450 mJ/10 Hz) is required for efficient debonding. CONCLUSIONS: There are differences regarding transmission rates between ceramic types for the Er:YAG laser light and additionally depending on the type of ceramic different energy settings should be used for adequate debonding. Based on our in-vitro experiments we recommend 2.5 W for zirconium-reinforced lithium silicate and zirconium dioxide and 4.5 W for lithium disilicate and feldspatic ceramic. Transmission rates of different ceramic types and varying influences of thicknesses and bonding materials should be considered to adjust the laser parameters during laser debonding of adhesively luted all-ceramic restorations.

The effect of combination treatment of CO2-laser irradiation and tetracalcium phosphate/dicalcium phosphate anhydrate on dentinal tubules blockage: an in vitro study.

The aim of this study was the evaluation of the in vitro efficacy of a carbon dioxide (CO2) laser, a tetracalcium phosphate/dicalcium phosphate anhydrate (TP/DP) desensitizer and the combination of the desensitizer and additional CO2 laser irradiation as a treatment modality for cervical dentin hypersensitivity. A total of 48 dental specimens, prepared from extracted human premolars and molars, were divided into four groups: a control group, a TP/DP desensitizer paste group, a CO2 laser (10.600-nm wavelength) group, and a paste and laser group. The specimens were coated with nail varnish except in the marked area and were then immersed in 2% methylene blue dye for 1 h. The specimens were then washed, dried, and cut longitudinally. Thereafter, photos of 40 dentin specimens were taken and evaluated. The area of penetration was assessed and reported as percentage of the dentin surface area. Additionally eight dental specimens were examined with the aid of a scanning electron microscope and evaluated. Significant differences in the penetration depth were found for all experimental groups compared to the control group. The lowest penetration area was detected in the paste-laser group (16.5%), followed by the laser (23.7%), the paste (48.5%), and the control group (86.2%). The combined treatment of the CO2 laser and a TP/DP desensitizer was efficient in sealing the dentinal surface and could be a treatment option for cervical dentin hypersensitivity.

Influence of Laser Irradiation Settings, during Diode-Assisted Endodontics, on the Intraradicular Adhesion of Self-Etch and Self-Curing Luting Cement during Restoration-An Ex Vivo Study.

Background: Diode-assisted endodontics is nowadays utilized for pulp space disinfection, but little is known on the bonding potential of this lased root dentin when the tooth is restored with an intracanal polymer post. Objectives: to investigate the influence of diode laser irradiation settings, in laser-assisted endodontics, on the intraradicular bonding of composite materials. Methods: Sixteen two-rooted, maxillary first premolars were collected, prepared up to F4 (Protaper Universal. Dentsply-Maillefer, Ballaigues, Switzerland), and randomly assigned in two groups: group A (chopped mode or short pulse), diode irradiated according to protocol, pulse 25 ms, power 2.5 W, and group B (microchopped mode or ultrashort pulse), pulse 25 µs, peak power 12 W (both groups GentleRay. KaVo Dental, Biberach an der Riss, Germany). Buccal canals were irradiated, palatal ones served as controls. Canals were then obturated, post space was created in all canals, and quartz-fiber posts (ICE light Danville. Danville Materials, San Ramon, CA, USA) were cemented by self-etch self-curing cement (Max Cem Elite. Kerr, West Collins Orange, CA, USA) (Max Cem Elite. Kerr, Brea, CA, USA). A week later, teeth were sectioned horizontally in 1 mm increments. Push-out test was conducted in a Zwick testing machine (Zwick Roell, Ulm, Germany) at 1 mm/min speed, and the force required to dislodge the post from each specimen (F-max) was recorded. Weibull regression models were applied for statistical analyses. Results: Differences in F-max by group (control vs. chopped mode vs. microchopped mode) and height (meaning the apical-to-coronal position of each specimen along the root) were statistically significant (p < 0.05 in all cases). Conclusions: Short pulses (or chopped mode) had a profound positive effect on the quality of intraradicular bonding, while Ultrashort pulses (or microchopped mode) affected it negatively. In addition, apically positioned bonding proved weaker compared with more coronally located specimens.

Impact of Implant Surface Material and Microscale Roughness on the Initial Attachment and Proliferation of Primary Human Gingival Fibroblasts.

Due to the rising demand for zirconia (Zr) based implant systems, it is important to understand the impact of Zr and titanium (Ti) implants and particularly their topography on soft tissue healing. As human gingival fibroblasts (hGFs) are the predominant cells in peri-implant soft tissue, we focused on examining the effect of implant material and surface roughness on hGFs' initial attachment, growth and the expression of proteins involved in the focal adhesion. hGFs isolated from eight healthy donors were cultured on the following surfaces: smooth titanium machined surface (TiM), smooth zirconia machined surface (ZrM), moderately rough titanium surface (SLA), or moderately rough zirconia surface (ZLA) for up to 14 days. The initial attachment of hGFs was evaluated by scanning electron microscopy. Cell proliferation/viability was assessed by cell counting kit 8. Focal adhesion and cytoskeleton were visualized by a focal adhesion staining kit. The gene expression of focal adhesion kinase (FAK), α-smooth muscle actin (α-SMA), and integrin subunits ITG-ß1, ITG-ß4, ITG-α4, ITG-α5, ITG-α6, was evaluated by qPCR. Cell proliferation/viability was slightly decreased by moderately rough surfaces, whereas no effect of surface material was observed. Cell morphology was strikingly different between differently treated surfaces: on machined surfaces, cells had elongated morphology and were attached along the grooves, whereas on moderately rough surfaces, cells were randomly attached. Surface roughness had a more pronounced effect on the gene expression compared to the surface material. The expression of FAK, α-SMA, ITG-ß4, ITG-α5, and ITG-α6 was enhanced by moderately rough surfaces compared to smooth surfaces. Within the limitations of this in vitro study, it can be concluded that the behavior of primary hGFs is primarily affected by surface structure, whereas no apparent advantage of Zr over Ti could be observed.

Effects of Er:YAG laser irradiation of different titanium surfaces on osteoblast response.

The aim of this in vitro study was to evaluate the effects of erbium-doped yttrium aluminum garnet (Er:YAG) laser irradiation on titanium surface topography and the proliferation and differentiation of osteoblasts using standard clinical treatment settings. Er:YAG laser irradiation at two levels ((1): 160 mJ, pulse at 20 Hz; (2): 80 mJ, pulse at 20 Hz) was applied to moderately rough and smooth titanium disks before MG-63 osteoblast-like cells were cultured on these surfaces. Titanium surface and cell morphology were observed by scanning electron microscopy. Cell proliferation/viability was measured by CCK-8 test. Gene expression of alkaline phosphatase (ALP), osteocalcin (OC), osteoprotegerin (OPG), receptor activator of nuclear factor kappa-B ligand (RANKL), and collagen type 1 was measured by qPCR, and OPG and OC protein production was determined by enzyme-linked immunosorbent assay. Treatment with Er:YAG laser at 160 mJ/20 Hz markedly caused heat-induced fusion of titanium and cell condensation on moderately rough surfaces, but not in smooth surfaces. MG-63 proliferation/viability decreased after 5 days in moderately rough surfaces. The expression of ALP, OC, OPG, and collagen type 1 was unaffected by laser treatment at 160 mJ/20. Laser irradiation at 80 mJ/20 Hz enhanced RANKL gene expression after 5 days in moderately rough surfaces. Study results suggest that Er:YAG laser irradiation at clinically relevant setting has no essential effect on osteogenic gene and protein expression of osteoblasts. However, surface structure, cell attachment, and proliferation are influenced by both treatment protocols, which implies that caution should be taken in the clinical treatment of peri-implant diseases when Er:YAG laser is used.

The impact of 3D-printed LAY-FOMM 40 and LAY-FOMM 60 on L929 cells and human oral fibroblasts.

OBJECTIVES: LAY-FOMM is a promising material for FDA-approved Fused Deposition Modeling (FDM) applications in drug delivery. Here we investigated the impact on oral cells. MATERIALS AND METHODS: We evaluated the impact of 3D-printed LAY-FOMM 40, LAY-FOMM 60, and biocompatible polylactic acid (PLA) on the activity of murine L929 cells, gingival fibroblasts (GF), and periodontal ligament fibroblasts (PDLF) using indirect (samples on cells), direct monolayer culture models (cells on samples), and direct spheroid cultures with resazurin-based toxicity assay, confirmed by MTT and Live-dead staining. The surface topography was evaluated with scanning electron microscopy. RESULTS: The materials LAY-FOMM 40 and LAY-FOMM 60 led to a reduction in resazurin conversion in L929 cells, GF, and PDLF, higher than the impact of PLA in indirect and direct culture models. Fewer vital cells were found in the presence of LAY-FOMM 40 and 60 than PLA, in the staining in both models. In the direct model, LAY-FOMM 40 and PLA showed less impact on viability in the resazurin-based toxicity assay than in the indirect model. Spheroid microtissues showed a reduction of cell activity of GF and PDLF with LAY-FOMM 40 and 60. CONCLUSION: Overall, we found that LAY-FOMM 40 and LAY-FOMM 60 can reduce the activity of L292 and oral cells. Based on the results from the PLA samples, the direct model seems more reliable than the indirect model. CLINICAL RELEVANCE: A material modification is desired in terms of biocompatibility as it can mask the effect of drugs and interfere with the function of the 3D-printed device.

Behaviour of Human Oral Epithelial Cells Grown on Invisalign® SmartTrack® Material.

Invisalign aligners have been widely used to correct malocclusions, but their effect on oral cells is poorly known. Previous research evaluated the impact of aligners' eluates on various cells, but the cell behavior in direct contact with aligners is not yet studied. In the present study, we seeded oral epithelial cells (cell line Ca9-22) directly on Invisalign SmartTrack material. This material is composed of polyurethane and co-polyester and exhibit better mechanical characteristics compared to the predecessor. Cell morphology and behavior were investigated by scanning electron microscopy and an optical cell moves analyzer. The effect of aligners on cell proliferation/viability was assessed by cell-counting kit (CCK)-8 and 3,4,5-dimethylthiazol-2-yl-2,5-diphenyl tetrazolium bromide (MTT) assay and live/dead staining. The expression of inflammatory markers and proteins involved in epithelial barrier function was measured by qPCR. Cells formed cluster-like structures on aligners. The proliferation/viability of cells growing on aligners was significantly lower (p < 0.05) compared to those growing on tissue culture plastic (TCP). Live/dead staining revealed a rare occurrence of dead cells on aligners. The gene expression level of all inflammatory markers in cells grown on aligners' surfaces was significantly increased (p < 0.05) compared to cells grown on TCP after two days. Gene expression levels of the proteins involved in barrier function significantly increased (p < 0.05) on aligners' surfaces after two and seven days of culture. Aligners' material exhibits no cytotoxic effect on oral epithelial cells, but alters their behavior and the expression of proteins involved in the inflammatory response, and barrier function. The clinical relevance of these effects has still to be established.

In vitro biocompatibility of biohybrid polymers membrane evaluated in human gingival fibroblasts.

The biohybrid polymer membrane (BHM) is a new biomaterial designed for the treatment of soft periodontal tissue defects. We aimed to evaluate the in vitro biocompatibility of the membrane in human gingival fibroblasts and the capability to induce cell adhesion, migration, differentiation and improving the production of the extracellular matrix. BHM and Mucograft® collagen matrix (MCM) membranes were punched into 6 mm diameter round discs and placed in 96-well plates. Human primary gingival fibroblasts were seeded on the membranes or tissue culture plastic (TCP) serving as the control. Cell proliferation/viability and morphology were evaluated after 3, 7, and 14 days of culture by cell counting kit (CCK)-8 assay and scanning electron microscopy, respectively. Additionally, the gene expression of transforming growth factor (TGF)-ß1, focal adhesion kinase (FAK), collagen type 1 (Col1), alpha-smooth muscle actin (α-SMA), and fibroblasts growth factor (FGF)-2 was analyzed at 3, 7, and 14 days of culture by qPCR. Cell proliferation on BHM was significantly higher than on MCM and similar to TCP. Gene expression of TGF-ß1, FAK, Col1, and α-SMA were significantly increased on BHM compared to TCP at most investigated time points. However, the gene expression of FGF-2 was significantly decreased on BHM at Day 7 and recovered at Day 14 to the levels similar to TCP. The finding of this study showed that BHM is superior for gingival fibroblasts in terms of adhesion, proliferation, and gene expression, suggesting that this membrane may promote the healing of soft periodontal tissue.

The response of periodontal cells to kaolinite.

OBJECTIVES: The impact of kaolinite on human periodontal cells is yet unknown. The aim of the study was to assess the response of human periodontal cells to kaolinite. METHODS: Human periodontal cells were treated with kaolinite at reducing concentrations from 30 to 0.0015 mg/mL and with conditioned medium, which was depleted of kaolinite. Cell viability was evaluated with a resazurin-based toxicity assay, Live-Dead staining, and MTT assay and staining. The pro-angiogenic factors vascular endothelial growth factor (VEGF) and interleukin (IL)-6 and IL-8 were quantified via ELISA in periodontal fibroblasts. L-929, a standard cell-line used for cytotoxicity studies, served as control cell line. Composition of kaolinite was verified using energy-dispersive X-ray spectroscopy. RESULTS: Kaolinite in suspension but not in conditioned medium impaired cell viability dose-dependently. VEGF, IL-6, and IL-8 production was not substantially modulated by kaolinite or the conditioned medium in periodontal cells. CONCLUSION: Overall, kaolinite can decrease cell viability dose-dependently while conditioned medium showed no toxic effect. No pronounced impact of kaolinite on VEGF, IL-6, and IL-8 production was observed. This study provided first insights into the impact of kaolinite on human periodontal cells thereby inferring to the basis for the evaluation of kaolinite as a carrier in regenerative dentistry. CLINICAL RELEVANCE: Kaolinite, a clay mineral, is successfully used in medicine due to its favorable properties. Also, applications in conservative dentistry are described. However, the response of oral cells to kaolinite is still unclear. Here, we assessed the impact of kaolinite on human periodontal cells.

Titanium dioxide-based scanning powder can modulate cell activity of oral soft tissue - Insights from in vitro studies with L929 cells and periodontal fibroblasts.

PURPOSE: To reveal the impact of titanium dioxide-based scanning powder for intraoral digital impression on the biological activity of oral fibroblasts. METHODS: Murine L929 cells and human periodontal ligament (PDLF) and gingival fibroblasts (GF) were treated with ten-fold serial dilutions of scanning powder and the corresponding conditioned medium (filtrate of overnight incubation of powder in medium) starting with 30mg/ml. Bicinchoninic acid protein assay, formazan- and resazurin-based toxicity assays, live/dead and annexin V/propidium iodide (PI) staining and immunoassays for interleukin (IL)-6 and IL-8 were performed. Powder composition was analyzed using energy dispersive X-ray spectroscopy (EDS). RESULTS: Formazan and resazurin conversion was lesser in L929 cells than PDLF and GF in the presence of scanning powder. Induction of cell death was caused by 30mg/ml of powder in L929 cells but not in PDLF and GF. No pronounced impact of the conditioned medium was seen in cytotoxicity assays or live/dead-, and annexin V/PI staining. In PDLF and GF IL-6 expression was increased by the powder, while there was a decrease in IL-8. Powder particles did not deplete protein from medium. EDS showed a heterogeneous mixture consisting predominantly of titanium dioxide. CONCLUSIONS: Scanning powder decreased cell activity and induced cell death in L929 cells at high concentrations. Human oral fibroblasts showed an increase in IL-6 levels but more resistance to the cytotoxicity of the powder. Within the limitations of an in vitro study our results suggest that proper cleaning after scanning is of clinical relevance to avoid potential unwanted effects of the powder.

In vitro evaluation of experimental light activated gels for tooth bleaching.

Dental bleaching is an important part of aesthetic dentistry. Various strategies have been created to enhance the bleaching efficacy. As one such strategy, light-activated nanoparticles that enable localized generation of reactive oxygen species have been developed. Here, we evaluated the cellular response to experimental gels containing these materials in in vitro models. L-929 cells, 3T3 cells, and gingival fibroblasts were exposed to the gels at 50%, 10%, 2%, 0.4%, 0.08%, 0.016%, and 0.0032%. The gels contained TiO2/Ag nanoparticles, TiO2 nanoparticles, hydrogen peroxide (6% hydrogen peroxide), or no added component and were tested with and without exposure to light. Cells were exposed to gels for 24 h or for 30 min. The latter case mimics the clinical situation of a short bleaching gel exposure. Metabolic activity and cell viability were evaluated with MTT and neutral red assays, respectively. We found a dose-dependent reduction of formazan formation and neutral red staining with gels containing TiO2/Ag nanoparticles or TiO2 nanoparticles in the 24 h setting with and without illumination. The strongest reduction, which was not dose-dependent in the evaluated concentrations, was found for the gel containing hydrogen peroxide. Gels with TiO2 nanoparticles showed a similar response to gel without particles. TiO2/Ag gel showed a slightly higher impact. When the gels were removed by rinsing after 30 min of exposure without light illumination, gel containing TiO2/Ag nanoparticles showed a stronger reduction of formazan formation and neutral red staining than gel containing TiO2 particles. Exposure of cells for 30 min under illumination and consequent rinsing off the gels also showed that Ag-containing particles can have a higher impact on the metabolic activity and viability than particles from TiO2. Overall our results show that experimental bleaching gels containing TiO2/Ag or TiO2 nanoparticles are less cytotoxic than hydrogen peroxide-containing gel. When gels are removed, gel containing TiO2/Ag particles exhibit a stronger reduction of metabolic activity and viability than the gel containing TiO2.

Evaluation of Resins for Stereolithographic 3D-Printed Surgical Guides: The Response of L929 Cells and Human Gingival Fibroblasts.

Additive manufacturing is becoming increasingly important in dentistry for the production of surgical guides. The development of cost-effective desktop stereolithography (SLA) printing systems and the corresponding resins makes this novel technique accessible to dental offices and dental laboratories. The aim of the study was to reveal the response of soft tissue cells to Clear and Dental SG resins used in desktop SLA printing systems at different stages of processing. Cell activity of L929 cells and gingival fibroblasts (GF) in response to the materials was examined in indirect and direct monolayer culture models and a direct spheroid culture model based on MTT, resazurin-based toxicity assays, and live-dead staining. Overall we found that the impact of Clear and Dental SG resins on L929 and GF depends on the processing stage of the materials. Liquid Clear resin induced a stronger reduction of cell activity compared to Dental SG resin. Printing and postcuring reduced the impact on cell activity and viability. As in-house 3D printing for surgical guides is getting integrated in the digital workflow, our data suggest that careful adherence to processing guidelines-especially postcuring-is of clinical relevance.

Effect of enamel matrix derivative on the angiogenic behaviors of human umbilical vein endothelial cells on different titanium surfaces.

Angiogenesis play a crucial role in the regeneration of hard and soft tissue around dental titanium (Ti) implant. Enamel matrix derivative (EMD) promotes tissue regeneration and stimulates angiogenesis but its effect on the angiogenesis on Ti surfaces was never investigated. The effect of EMD on the angiogenic activity of endothelial cells cultured on pre-treated smooth Ti (PT), acidetched (A), coarse-grit blasted and acid-etched (SLA) surfaces and tissue culture plastic (TCP) in the presence or absence of EMD was investigated. EMD inhibited the proliferation/viability of human umbilical vein endothelial cells (HUVECs) growing on A and SLA Ti surfaces. EMD induced an increase in the expression of all these genes in HUVECs grown on SLA surface but not on other surfaces. Summarizing, our data show that EMD influences proliferation and expression of angiogenesis associated gene in HUVECs grown on moderately rough SLA surfaces, suggesting that EMD might promote angiogenesis following implantation.