Cytotoxicity and genotoxicity of various types of endodontic sealers in Chinese hamster ovary (CHO-K1) cells.

This study aimed to evaluate the cytotoxicity and genotoxicity of five endodontic sealers (AH Plus, MTA Fillapex, Endoseal MTA, Sealapex, and Zinc oxide eugenol) in Chinese hamster ovary cells. Cytotoxicity was evaluated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay to check cell viability at 1, 3, and 7 days. Genotoxicity was assessed by cytokinesis-block micronucleus, single-cell gel electrophoresis, and γH2AX immunofluorescence assays. Cell viability of all endodontic sealers, except Endoseal MTA, on day 1 was less than 100%. Endoseal MTA showed the highest cell viability on day 7. AH Plus and Endoseal MTA showed less DNA damage than other sealers. After complete setting, AH Plus and Endoseal MTA showed low genotoxicity, which could reduce DNA damage in periapical cells, making them suitable as endodontic sealers.

[National Petition Analysis Related to Nursing: Text Network Analysis and Topic Modeling].

PURPOSE: This study aimed to identify the main keyword, network structure, and main topics of the national petition related to "nursing" in South Korea. METHODS: Data were gathered from petitions related to the national petition in Korea Blue House related to the topic "nursing" or "nurse" from August 17, 2017, to May 9, 2022. A total of 5,154 petitions were searched, and 995 were selected for the final analysis. Text network analysis and topic modeling were analyzed using the Netminer 4.5.0 program. RESULTS: Regarding network characteristics, a density of 0.03, an average degree of 144.483, and an average distance of 1.943 were found. Compared to results of degree centrality and betweenness centrality, keywords such as "work environment," "nursing university," "license," and "education" appeared typically in the eigenvector centrality analysis. Topic modeling derived four topics: (1) "Improving the working environment and dealing with nursing professionals," (2) "requesting investigation and punishment related to medical accidents," (3) "requiring clear role regulation and legislation of medical and nonmedical professions," and (4) "demanding improvement of healthcare-related systems and services." CONCLUSION: This is the first study to analyze Korea's national petitions in the field of nursing. This study's results confirmed both the internal needs and external demands for nurses in South Korea. Policies and laws that reflect these results should be developed.

Effects of the cathepsin K inhibitor with mineral trioxide aggregate cements on osteoclastic activity.

OBJECTIVES: Root resorption is an unexpected complication after replantation procedures. Combining anti-osteoclastic medicaments with retrograde root filling materials may avert this resorptive activity. The purpose of this study was to assess effects of a cathepsin K inhibitor with calcium silicate-based cements on osteoclastic activity. METHODS: MC3T3-E1 cells were cultured for biocompatibility analyses. RAW 264.7 cells were cultured in the presence of the receptor activator of nuclear factor-kappa B and lipopolysaccharide, followed by treatment with Biodentine (BIOD) or ProRoot MTA with or without medicaments (Odanacatib [ODN], a cathepsin inhibitor and alendronate, a bisphosphonate). After drug treatment, the cell counting kit-8 assay and Alizarin red staining were performed to evaluate biocompatibility in MC3T3-E1 cells. Reverse-transcription polymerase chain reaction, tartrate-resistant acid phosphatase (TRAP) staining and enzyme-linked immunosorbent assays were performed in RAW 264.7 cells to determine the expression levels of inflammatory cytokines, interleukin (IL)-1ß, IL-6, tumor necrosis factor-α (TNF-α) and prostaglandin E2 (PGE2). Data were analyzed by one-way analysis of variance and Tukey's post hoc test (p < 0.05). RESULTS: Biocompatibility results showed that there were no significant differences among any of the groups. RAW 264.7 cells treated with BIOD and ODN showed the lowest levels of TNF-α and PGE2. Treatments with BIOD + ODN were more potent suppressors of inflammatory cytokine expression (p < 0.05). CONCLUSION: The cathepsin K inhibitor with calcium silicate-based cement inhibits osteoclastic activity. This may have clinical application in preventing inflammatory root resorption in replanted teeth.

Effect of ProRoot MTA® and Biodentine® on osteoclastic differentiation and activity of mouse bone marrow macrophages.

OBJECTIVES: This investigation aimed to assess the differentiation inhibitory effects of ProRoot MTA® (PMTA) and Biodentine® (BIOD) on osteoclasts originated from murine bone marrow macrophages (BMMs) and compare these effects with those of alendronate (ALD). MATERIALS AND METHODS: Mouse BMMs were cultured to differentiate into osteoclasts with macrophage colony-stimulating factor and receptor activator of NF-κB (RANKL), treated with lipopolysaccharide. After application with PMTA, BIOD, or ALD, cell toxicities were examined using WST-1 assay kit, and RANKL-induced osteoclast differentiation and activities were determined by resorption pit formation assay and tartrate-resistant acid phosphate (TRAP) staining. The mRNA levels of osteoclast activity-related genes were detected with quantitative real time polymerase chain reaction. Expressions of molecular signaling pathways were assessed by western blot. All data were statistically analyzed with one-way ANOVA and Tukey's post-hoc test (p<0.05). RESULTS: Mouse BMMs applied with PMTA, BIOD, or ALD showed highly reduced levels of TRAP-positive osteoclasts. The BIOD treated specimens suppressed mRNA expressions of cathepsin K, TRAP, and c-Fos. Nonetheless, it showed a lower effect than PMTA or ALD applications. Compared with ALD, PMTA and BIOD decreased RANKL-mediated phosphorylation of ERK1/2 and IκBα. CONCLUSIONS: PMTA and BIOD showed the inhibitory effect on osteoclast differentiation and activities similar to that of ALD through IκB phosphorylation and suppression of ERK signaling pathways.

Effect of ProRoot MTA® and Biodentine® on osteoclastic differentiation and activity of mouse bone marrow macrophages

Abstract Objectives This investigation aimed to assess the differentiation inhibitory effects of ProRoot MTA® (PMTA) and Biodentine® (BIOD) on osteoclasts originated from murine bone marrow macrophages (BMMs) and compare these effects with those of alendronate (ALD). Materials and Methods Mouse BMMs were cultured to differentiate into osteoclasts with macrophage colony-stimulating factor and receptor activator of NF-κB (RANKL), treated with lipopolysaccharide. After application with PMTA, BIOD, or ALD, cell toxicities were examined using WST-1 assay kit, and RANKL-induced osteoclast differentiation and activities were determined by resorption pit formation assay and tartrate-resistant acid phosphate (TRAP) staining. The mRNA levels of osteoclast activity-related genes were detected with quantitative real time polymerase chain reaction. Expressions of molecular signaling pathways were assessed by western blot. All data were statistically analyzed with one-way ANOVA and Tukey's post-hoc test (p<0.05). Results Mouse BMMs applied with PMTA, BIOD, or ALD showed highly reduced levels of TRAP-positive osteoclasts. The BIOD treated specimens suppressed mRNA expressions of cathepsin K, TRAP, and c-Fos. Nonetheless, it showed a lower effect than PMTA or ALD applications. Compared with ALD, PMTA and BIOD decreased RANKL-mediated phosphorylation of ERK1/2 and IκBα. Conclusions PMTA and BIOD showed the inhibitory effect on osteoclast differentiation and activities similar to that of ALD through IκB phosphorylation and suppression of ERK signaling pathways.

The effect of cathepsin K inhibitor surface treatment on delayed tooth replantation in dogs.

BACKGROUND/AIM: Continuous research efforts have been focusing on promoting healing of delayed replantation of avulsed teeth. The aim of this in vivo study was to assess the effect of cathepsin K inhibitor surface treatment on delayed tooth replantation. MATERIALS AND METHODS: Thirty-four premolar roots of 4 beagle dogs were extracted, and conventional root canal treatment was performed. The canals were filled with gutta-percha and cement. All extracted teeth were air-dried for 1 hour. Teeth were divided into 2 groups according to the root surface treatment prior to replantation: control (n = 13) and cathepsin K inhibitor (n = 21). Treated teeth were replanted in their original sockets and stabilized with a resin-wire splint for 1 week. After 12 weeks, the dogs were euthanized and micro-computed tomography was performed. Tissues were then further processed as resin-embedded specimens stained with hematoxylin and eosin. In each evaluation, data were analyzed using the Mann-Whitney U test (P < .05). RESULTS: In the micro-computed tomography evaluation, inflammation scores of both groups were not statistically different (P > .05). In the histological evaluation, the mean proportions of inflammatory resorption and replacement resorption in the control group were similar to those in the cathepsin K inhibitor group (P > .05). CONCLUSIONS: Cathepsin K inhibitor did not demonstrate significant inhibitory effects on root resorption after delayed replantation in vivo.

Cytotoxicities and genotoxicities of cements based on calcium silicate and of dental formocresol.

Increasing interest is being paid to the toxicities of dental materials. The purpose of this study was to determine the cytotoxicities and genotoxicities of endodontic compounds to Chinese hamster ovary (CHO-K1) reproductive cells. Cultured CHO-K1 cells were treated with dental formocresol, two types of calcium hydroxide paste, and two types of mineral trioxide aggregate cement for 24h. A 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide assay was performed on each culture, and the micronucleus frequency was determined by performing a micronucleus assay. Alkaline comet assay and γ-H2AX immunofluorescence assay were used to detect DNA damage. Out of the five materials tested, only dental formocresol significantly increased DNA damage. The mineral trioxide aggregate cements based on calcium silicate were not found to be potentially genotoxic. The data suggest that dental formocresol should not be recommended for use in vital pulp therapy on young teeth.

Cytotoxicity and biocompatibility of Zirconia (Y-TZP) posts with various dental cements.

OBJECTIVES: Endodontically treated teeth with insufficient tooth structure are often restored with esthetic restorations. This study evaluated the cytotoxicity and biological effects of yttria partially stabilized zirconia (Y-TZP) blocks in combination with several dental cements. MATERIALS AND METHODS: Pairs of zirconia cylinders with medium alone or cemented with three types of dental cement including RelyX U200 (3M ESPE), FujiCEM 2 (GC), and Panavia F 2.0 (Kuraray) were incubated in medium for 14 days. The cytotoxicity of each supernatant was determined using 3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assays on L929 fibroblasts and MC3T3-E1 osteoblasts. The levels of interleukin-6 (IL-6) mRNA were evaluated by reverse transcription polymerase chain reaction (RT-PCR), and IL-6 protein was evaluated by enzyme-linked immunosorbent assays (ELISA). The data were analyzed using one-way ANOVA and Tukey post-hoc tests. A p < 0.05 was considered statistically significant. RESULTS: The MTT assays showed that MC3T3-E1 osteoblasts were more susceptible to dental cements than L929 fibroblasts. The resin based dental cements increased IL-6 expression in L929 cells, but reduced IL-6 expression in MC3T3-E1 cells. CONCLUSIONS: Zirconia alone or blocks cemented with dental cement showed acceptable biocompatibilities. The results showed resin-modified glass-ionomer based cement less produced inflammatory cytokines than other self-adhesive resin-based cements. Furthermore, osteoblasts were more susceptible than fibroblasts to the biological effects of dental cement.

The effect of cathepsin K inhibitor on osteoclastic activity compared to alendronate and enamel matrix protein.

BACKGROUND/AIM: There have been several attempts to treat delayed replantation with agents that inhibit root resorption. The purpose of this study was to assess the effectiveness of cathepsin K inhibitor in inhibiting osteoclastic activity compared to that of alendronate and enamel matrix protein. MATERIALS AND METHODS: Murine RAW 264.7 cells were cultured in the presence of the receptor activator of NF-kB and lipopolysaccharide, followed by treatment with odanacatib, alendronate, or Emdogain at various concentrations. After drug treatment, an MTT assay was performed to evaluate cytotoxicity, while reverse transcription polymerase chain reaction and enzyme-linked immunosorbent assays were performed to determine the expression levels of interleukin-1ß, interleukin-6, prostaglandin E2, and tumor necrosis factor-α. Data were analyzed by one-way anova and Tukey's post-hoc test (P < 0.05). RESULTS: Of all tested agents, Emdogain resulted in the least cytotoxicity on RAW 264.7 cells, while 10(-9) M odanacatib had the largest suppressive effects on the expression levels of inflammatory cytokines. CONCLUSIONS: Odanacatib inhibits osteoclastic activity, showing the possibility as a treatment agent for delayed replantation of avulsed teeth.

The effects of bone morphogenetic protein-2 and enamel matrix derivative on the bioactivity of mineral trioxide aggregate in MC3T3-E1cells.

OBJECTIVES: The effects of bone morphogenetic protein-2 (BMP-2) and enamel matrix derivative (EMD) respectively with mineral trioxide aggregate (MTA) on hard tissue regeneration have been investigated in previous studies. This study aimed to compare the osteogenic effects of MTA/BMP-2 and MTA/EMD treatment in MC3T3-E1 cells. MATERIALS AND METHODS: MC3T3-E1 cells were treated with MTA (ProRoot, Dentsply), BMP-2 (R&D Systems), EMD (Emdogain, Straumann) separately and MTA/BMP-2 or MTA/EMD combination. Mineralization was evaluated by staining the calcium deposits with alkaline phosphatase (ALP, Sigma-Aldrich) and Alizarin red (Sigma-Aldrich). The effects on the osteoblast differentiation were evaluated by the expressions of osteogenic markers, including ALP, bone sialoprotein (BSP), osteocalcin (OCN), osteopontin (OPN) and osteonectin (OSN), as determined by reverse-transcription polymerase chain reaction analysis (RT-PCR, AccuPower PCR, Bioneer). RESULTS: Mineralization increased in the BMP-2 and MTA/BMP-2 groups and increased to a lesser extent in the MTA/EMD group but appeared to decrease in the MTA-only group based on Alizarin red staining. ALP expression largely decreased in the EMD and MTA/EMD groups based on ALP staining. In the MTA/BMP-2 group, mRNA expression of OPN on day 3 and BSP and OCN on day 7 significantly increased. In the MTA/EMD group, OSN and OCN gene expression significantly increased on day 7, whereas ALP expression decreased on days 3 and 7 (p < 0.05). CONCLUSIONS: These results suggest the MTA/BMP-2 combination promoted more rapid differentiation in MC3T3-E1 cells than did MTA/EMD during the early mineralization period.

Comparison of the biological properties of ProRoot MTA, OrthoMTA, and Endocem MTA cements.

INTRODUCTION: OrthoMTA (BioMTA, Seoul, Korea) and Endocem MTA (Maruchi, Wonju-si, Korea) were recently developed to overcome the disadvantages of ProRoot MTA (Dentsply, Tulsa, OK). This study aimed to compare the biological properties of OrthoMTA and Endocem MTA with those of ProRoot MTA using the preosteoblastlike cell line MC3T3-E1. METHODS: The setting times of calcium silicate-based cements (CSCs) and their effects on the pH of distilled water during storage were determined according to ISO standards. MC3T3-E1 cells were cultured with ProRoot MTA, OrthoMTA, and Endocem MTA. The viability of the cells was assessed using the Cell Counting Kit-8 assay (Dojindo Laboratory, Kumamoto, Japan) on the supernatants of CSCs, and the cells' osteopontin production was determined by an enzyme-linked immunosorbent assay on a culture with the materials on days 3 and 7 of incubation. RESULTS: Endocem MTA exhibited a significantly shorter setting time (15.3 ± 0.5 minutes) than did ProRoot MTA and OrthoMTA (318.0 ± 56.0 and 324.3 ± 2.1 minutes, P < .05). Additionally, all CSCs caused their storage water to become highly alkaline after 7 days. OrthoMTA was significantly more cytotoxic than ProRoot and Endocem MTA (P < .05). ProRoot MTA induced significantly more OPN production than OrthoMTA and Endocem MTA on both days 3 and 7 (P < .05). CONCLUSIONS: ProRoot MTA appeared to be superior to OrthoMTA and Endocem MTA in terms of biological properties although Endocem MTA exhibited the shortest setting time and presented lower cytotoxicity with osteoblastlike cells.

Evaluation of the rat tissue reaction to experimental new resin cement and mineral trioxide aggregate cement.

OBJECTIVES: New resin cement (NRC) has been developed as a root repairing material and the material is composed of organic resin matrix and inorganic powders. The aim of this study was to compare the rat subcutaneous tissue response to NRC and mineral trioxide aggregate (MTA) cement and to investigate the tissue toxicity of both materials. MATERIALS AND METHODS: Sixty rats received two polyethylene tube-implants in dorsal subcutaneous regions, MTA and NRC specimens. Twenty rats were sacrificed respectively at 1, 4 and 8 wk after implantation and sectioned to 5 µm thickness and stained with Hematoxylin-Eosin (H-E) or von-Kossa staining. The condition of tissue adjacent to the implanted materials and the extent of inflammation to each implant were evaluated by two examiners who were unaware of the type of implanted materials in the tissues. Data were statistically analyzed with paired t-test (p < 0.05). RESULTS: In specimens implanted with both NRC and MTA, severe inflammatory reactions were present at one wk, which decreased with time. At eighth wk, MTA implanted tissue showed mild inflammatory reaction, while there were moderate inflammatory reactions in NRC implanted tissue, respectively. In NRC group, von-Kossa staining showed more calcification materials than MTA group at eighth wk. CONCLUSIONS: It was concluded that the calcium reservoir capability of NRC may contribute to mineralization of the tissues.

Accuracy of Root ZX in teeth with simulated root perforation in the presence of gel or liquid type endodontic irrigant.

OBJECTIVES: To evaluate the accuracy of the Root ZX in teeth with simulated root perforation in the presence of gel or liquid type endodontic irrigants, such as saline, 5.25% sodium hypochlorite (NaOCl), 2% chlorhexidine liquid, 2% chlorhexidine gel, and RC-Prep, and also to determine the electrical conductivities of these endodontic irrigants. MATERIALS AND METHODS: A root perforation was simulated on twenty freshly extracted teeth by means of a small perforation made on the proximal surface of the root at 4 mm from the anatomic apex. Root ZX was used to locate root perforation and measure the electronic working lengths. The results obtained were compared with the actual working length (AWL) and the actual location of perforations (AP), allowing tolerances of 0.5 or 1.0 mm. Measurements within these limits were considered as acceptable. Chi-square test or the Fisher's exact test was used to evaluate significance. Electrical conductivities of each irrigant were also measured with an electrical conductivity tester. RESULTS: The accuracies of the Root ZX in perforated teeth were significantly different between liquid types (saline, NaOCl) and gel types (chlorhexidine gel, RC-Prep). The accuracies of electronic working lengths in perforated teeth were higher in gel types than in liquid types. The accuracy in locating root perforation was higher in liquid types than gel types. 5.25% NaOCl had the highest electrical conductivity, whereas 2% chlorhexidine gel and RC-Prep gel had the lowest electrical conductivities among the five irrigants. CONCLUSIONS: Different canal irrigants with different electrical conductivities may affect the accuracy of the Root ZX in perforated teeth.

Evaluation of physical and biologic properties of the mixture of mineral trioxide aggregate and 4-META/MMA-TBB resin.

OBJECTIVE: This study examined whether 4-methacryloxyethyl trimellitate anhydride/methylmethacrylate-tri-n-butyl borane (4-META/MMA-TBB) resin can be used with mineral trioxide aggregate (MTA) to overcome MTA's shortcomings. The biologic reactions of the mixture of MTA powder and 4-META/MMA-TBB resin (MTA/4-META) and its potential in clinical applications were also investigated. STUDY DESIGN: MTA powder was mixed with 4-META/MMA-TBB resin instead of water at the appropriate proportions determined by a series of studies prior to this experiment. MTA powder mixed with sterile water was used as control. The setting time, compressive strength, pH, and dye leakage of MTA/4-META and MTA were assessed by Gilmore apparatus, universal mechanical testing machine, pH meter, and methylene blue penetration method, respectively. Cytotoxicity also was evaluated by MTT assay with MC3T3-E1 cells. RESULTS: The setting time of MTA/4-META was significantly lower than that of MTA: 11.2 ± 0.8 minutes versus 318 ± 56.0 minutes, respectively (P < .05). The mean compressive strength of MTA/4-META after 24 hours was significantly higher than that of MTA: 57.4 ± 11.6 MPa versus 18.7 ± 3.0 MPa, respectively (P < .05). MTA/4-META showed significantly less leakage than MTA (P < .05). The initial pHs for MTA and MTA/4-META at 2 hours were 10.73 ± 0.95 and 10.08 ± 0.13, respectively, and reached plateaus of 10.92 ± 0.31 and 10.54 ± 0.39 at 24 hours, respectively. The pH of MTA was higher than that of MTA/4-META in the entire period, but the differences were only significant up to 48 hours (P < .05). MTA and MTA/4-META both showed no cytotoxicity. CONCLUSIONS: 4-META/MMA-TBB resin as a mixing vehicle of MTA powder can improve the setting and handling properties of MTA and may maintain or improve its other biophysical properties.

An interesting healing outcome of a replanted immature permanent tooth: a case report.

A case of an avulsed upper left central incisor that was replanted after 3 h in a 7-year-old girl is presented. The tooth showed signs of an acute periapical abscess at 2 weeks after replantation. Apexification with mineral trioxide aggregate (MTA) following application of calcium hydroxide was attempted. At 3-year and 6-month follow-up, the tooth was asymptomatic with adequate clinical function. The radiograph showed resolution of the periapical lesion and apparent radiopaque tissue under MTA plug resembling root end morphology.

Cytotoxicity of mineral trioxide aggregate (MTA) and bone morphogenetic protein 2 (BMP-2) and response of rat pulp to MTA and BMP-2.

OBJECTIVE: The aim of this study was to assess the cytotoxicity of mineral trioxide aggregate (MTA) and bone morphogenetic protein 2 (BMP-2) and the response of rat pulp tissue to MTA and BMP-2. STUDY DESIGN: For cytotoxicity studies, 1 g MTA was mixed with or without 1 microg of BMP-2 and allowed to set for 1, 24, 48, or 72 hours before addition of samples to 2-mL aliquots of culture medium. The viability of MG-63 cells was determined using the dimethyl-thiazol-diphenyltetrazolium bromide (MTT) assay. For animal studies, upper first molars from 32 Sprague-Dawley rats were used, the molars were exposed, and 1 g MTA cement was placed in the first molars. In left molars, 1 microg BMP-2 was additionally placed on exposed pulps with MTA. After 2 weeks and 7 weeks, rats were killed and histologic sections assessed by light microscopy. RESULTS: In MTT assay, the viability was higher in the MTA with BMP-2 group than in the MTA-only group up to 24 hours, but was not significantly different thereafter. In animal study, inflammation was higher in the MTA-only group than in the MTA with BMP group, although this difference did not attain statistical significance. CONCLUSIONS: The addition of BMP-2 had a beneficial effect in vitro, reducing the initial cytotoxicity of freshly mixed MTA. However, the pulp reaction to a combination of MTA and BMP-2 was not significantly better than use of MTA alone.

A new resin-bonded retrograde filling material.

OBJECTIVE: This study determined the physical properties and cytotoxicity of a novel root-end filling material (NRC). STUDY DESIGN: NRC is a powder and liquid system. The liquid is composed of hydroxyethylmethacrylate, benzoyl peroxide, toluidine, and toluenesulfinate. And the powder is made of calcium oxide, calcium silicate, and triphenylbismuth carbonate. The setting time, compressive strength, and pH change of NRC and gray and white mineral trioxide aggregate (MTA) were determined according to ISO standardization. MC3T3-E1 cells were cultured on NRC and white MTA for determining MTT scores. The absorbance of formazan was measured at 570 nm with a spectrophotometer. The MTT assay was performed in triplicate and repeated in 2 cultures. One-way analysis of variance was used to determine statistical differences in physical properties and MTT assay (P < .05). RESULTS: Mean setting time of materials tested were: NRC 12.5 +/- 0.3 minutes, gray MTA 345.5 +/- 96.2 minutes, and white MTA 318.0 +/- 56.0 minutes. After 24 hours, the mean compressive strengths were: NRC, 21.6 +/- 5.5 MPa, gray MTA: 7.7 +/- 3.3 MPa, and white MTA, 18.9 +/- 3.2 MPa. The pH of the test materials were: NRC 12.0, gray MTA 12.2, and white MTA 11.9. There were no statistically significant differences in compressive strength and pH between white MTA and NRC. The compressive strength of gray MTA was significantly lower than white MTA and NRC (P < .05). The setting time of NRC was significantly lower than white and gray MTA. In MTT assay, both NRC and white MTA were not cytotoxic to MC3T3-E1 cells. CONCLUSIONS: It was concluded that the setting time, compressive strength, pH, and initial biocompatibility results of NRC are favorable for a root-end filling material.