Effects of Enamel Matrix Derivative on Cell Spheroids Made of Stem Cells Obtained from the Gingiva on Osteogenic Differentiation.

Background and Objectives: A derivative of the enamel matrix was used to speed up periodontal regeneration, including the formation of new cementum, alveolar bone, and periodontal ligament. In this study, human gingiva-derived stem cell-derived cell spheroids were used to assess the effects of an enamel matrix derivative on cell viability, osteogenic differentiation, and mineralization. Materials and Methods: Human gingiva-derived stem cells were used to create spheroids, which were then coupled with unloaded control groups and an enamel matrix derivative at a final concentration of 2.7, 27, 270, and 2700 µg/mL. The morphological examination of the created stem cell spheroids took place on days 1, 3, 5, and 7. The Live/Dead Kit assay was used to determine the qualitative viability of cells on days 3 and 7. Using the Cell Counting Kit-8, the quantitative vitality of the cell spheroids was assessed on days 1, 3, and 5. On days 7 and 14, alkaline phosphatase activity assays and Alizarin Red S staining were carried out to examine the osteogenic differentiation of the cell spheroids. RUNX2 and COL1A1 expression levels on days 7 and 14 were determined using real-time polymerase chain reaction. Results: The added enamel matrix derivative at the tested concentrations did not significantly alter the morphology of the applied stem cells' well-formed spheroids on day 1. On days 3 and 7, the majority of the spheroids' cells fluoresced green while they were being cultivated. Alkaline phosphatase activity data revealed a substantial rise in the 2700 µg/mL group on day 7 when compared to the unloaded control (p < 0.05). On days 7 and 14, calcium deposits were distinctly seen in each group. In the 27 and 2700 µg/mL groups, the treatment with the enamel matrix derivative resulted in noticeably higher values for the Alizarin Red S staining (p < 0.05). qPCR results showed that adding an enamel matrix derivative to the culture of the 27 µg/mL group raised the level of RUNX2 mRNA expression. Conclusions: These results lead us to the conclusion that a derivative of the enamel matrix may be used to promote osteogenic differentiation in stem cell spheroids.

Bone Morphogenetic Protein-9 Promotes Osteogenic Differentiation and Mineralization in Human Stem-Cell-Derived Spheroids.

Background and Objectives: Alkaline phosphatase activity, mineralized matrix, and osteogenic-related gene expression have been shown to increase in response to bone morphogenetic protein-9 (BMP-9). In this study, spheroids derived from human gingival stem cells were used to determine the effects of BMP-9 on cell survival, osteogenesis, and mineralization. Materials and Methods: Human gingival stem cells were used to produce spheroids and then grown to concentrations of 0, 0.1, 1, 10, and 100 ng/mL with BMP-9. On days 1, 3, 5, and 7, morphological examination was carried out. A live/dead assay and Cell Counting Kit-8 was used to assess the vitality of cells. On days 7 and 14, alkaline phosphatase activity assays were carried out using a commercially available kit to examine the osteogenic differentiation of cell spheroids. Alizarin Red Staining was performed on the 7th and 14th days to evaluate mineralization, and RUNX2 and COL1A1 expression levels were evaluated on the 7th and 14th days using real-time polymerase chain reactions. Results: The BMP-9 added at the measured quantities did not appear to alter the shape of the well-formed spheroids produced by stem cells on day 1. In addition, treatment with BMP-9 at doses of 0, 0.1, 1, 10, or 100 ng/mL did not significantly alter cell diameter. Throughout the whole experimental process, viability was maintained. On day 14, the alkaline phosphatase activity in the groups dosed with 0.1, 1, 10, or 100 ng/mL was statistically higher than that in the unloaded control group (p < 0.05). According to qPCR data, the mRNA expression level of RUNX2 with 1 ng/mL dosing was higher on day 7 compared to that of the unloaded control group (p < 0.05). Conclusions: These findings suggest that BMP-9 can be employed to stimulate early osteogenic differentiation in stem cell spheroids.

Impact of 17ß-Estradiol on the Shape, Survival, Osteogenic Transformation, and mRNA Expression of Gingiva-Derived Stem Cell Spheroids.

Background and Objectives: Mesenchymal stem cells hold promise for tissue regeneration, given their robust growth and versatile differentiation capabilities. An analysis of bone marrow-sourced mesenchymal stem cell proliferation showed that 17ß-estradiol could enhance their growth. This study aims to investigate the influence of 17ß-estradiol on the shape, survival, osteogenic differentiation, and mineralization of human mesenchymal stem cells. Materials and Methods: Spheroids made from human gingiva-derived stem cells were cultivated with varying concentrations of 17ß-estradiol: 0, 0.01, 0.1, 1, and 10 nM. Morphology was assessed on days 1, 3, and 5. The live/dead kit assay was employed on day 3 for qualitative cell viability, while cell counting kit-8 was used for quantitative viability assessments on days 1, 3, and 5. To evaluate the osteogenic differentiation of the spheroids, a real-time polymerase chain reaction assessed the expressions of RUNX2 and COL1A1 on day 7. Results: The stem cells formed cohesive spheroids, and the inclusion of 17ß-estradiol did not noticeably alter their shape. The spheroid diameter remained consistent across concentrations of 0, 0.01, 0.1, 1, and 10 nM of 17ß-estradiol. However, cellular viability was boosted with the addition of 1 and 10 nM of 17ß-estradiol. The highest expression levels for RUNX2 and COL1A1 were observed with the introduction of 17ß-estradiol at 0.1 nM. Conclusions: In conclusion, from the results obtained, it can be inferred that 17ß-estradiol can be utilized for differentiating stem cell spheroids. Furthermore, the localized and controlled use, potentially through localized delivery systems or biomaterials, can be an area of active research. While 17ß-estradiol holds promise for enhancing stem cell applications, any clinical use requires a thorough understanding of its mechanisms, careful control of its dosage and delivery, and extensive testing to ensure safety and efficacy.

NELL-1 Increased the Osteogenic Differentiation and mRNA Expression of Spheroids Composed of Stem Cells.

Background and objectives: NELL-1 is a competent growth factor and it reported to target cells committed to the osteochondral lineage. The secreted, osteoinductive glycoproteins are reported to rheostatically control skeletal ossification. This study was performed to determine the effects of NELL-1 on spheroid morphology and cell viability and the promotion of osteogenic differentiation of stem cell spheroids. Materials and Methods: Cultures of stem cell spheroids of gingiva-derived stem cells were grown in the presence of NELL-1 at concentrations of 1, 10, 100, and 500 ng/mL. Evaluations of cell morphology were performed using a microscope, and cell viability was assessed using a two-color assay and Cell Counting Kit-8. Evaluation of the activity of alkaline phosphatase and calcium deposition assays involved anthraquinone dye assay to determine the level of osteogenic differentiation of cell spheroids treated with NELL-1. Real-time quantitative polymerase chain reaction (qPCR) was used to evaluate the expressions of RUNX2, BSP, OCN, COL1A1, and ß-actin mRNAs. Results: The applied stem cells produced well-formed spheroids, and the addition of NELL-1 at tested concentrations did not show any apparent changes in spheroid shape. There were no significant changes in diameter with addition of NELL-1 at 0, 1, 10, 100, and 500 ng/mL concentrations. The quantitative cell viability results derived on Days 1, 3, and 7 did not show significant disparities among groups (p > 0.05). There was statistically higher alkaline phosphatase activity in the 10 ng/mL group compared with the unloaded control on Day 7 (p < 0.05). A significant increase in anthraquinone dye staining was observed with the addition of NELL-1, and the highest value was noted at 10 ng/mL (p < 0.05). qPCR results demonstrated that the mRNA expression levels of RUNX2 and BSP were significantly increased when NELL-1 was added to the culture. Conclusions: Based on these findings, we conclude that NELL-1 can be applied for increased osteogenic differentiation of stem cell spheroids.

Dexamethasone downregulates SIRT1 and IL6 and upregulates EDN1 genes in stem cells derived from gingivae via the AGE/RAGE pathway.

OBJECTIVES: To evaluate the effects of dexamethasone on the aging of mesenchymal stem cells from human gingiva using next-generation sequencing. RESULTS: Four mRNAs were upregulated and 12 were downregulated when the results of dexamethasone at 24 h were compared with the control at 24 h. Expressions of SIRT1 and IL6 were decreased in dexamethasone at 24 h but expression of EDN1 was increased. CONCLUSIONS: Application of dexamethasone reduced the expression of SIRT1 and IL6 but enhanced the expression of EDN1 of stem cells.

Effects of Valproic Acid on Morphology, Proliferation, and Differentiation of Mesenchymal Stem Cells Derived From Human Gingival Tissue.

PURPOSE: Valproic acid (VPA), a histone deacetylase inhibitor, has been shown to affect cell growth and differentiation in various in vitro and in vivo models. The aim of this study is to evaluate the effects of VPA on viability and osteogenic differentiation of mesenchymal stem cells derived from the human gingival tissue. MATERIALS AND METHODS: Stem cells derived from the gingiva were grown in the presence of VPA at concentrations ranging from 0.125 to 8 mM. Cell morphology was assessed on days 3, 5, and 7, and cell proliferation was analyzed on the same days using a Cell Counting Kit-8 (CCK-8). Alizarin Red-S staining was used to assess differentiation of the stem cells. RESULTS: The control group showed a normal fibroblast morphology when cultured in growth media. The shape of cells in the 8 mM group was more flat than cells in other groups, and fewer cells were present. A statistically significant decrease in cell proliferation was seen in the 8 mM group. Results of Alizarin Red-S staining showed a significant decrease in mineralization in the 8 mM group. CONCLUSIONS: Taken together, this study demonstrated that VPA, at the tested concentrations, decreases the viability of stem cells derived from the human gingiva. The decreases in osteogenic differentiation were achieved via the decrease of Rux2 expression. The concentration and application time of VPA treatment should be meticulously controlled to minimize any detrimental effects.

Self-Assembly Enabled Printable Asymmetric Self-Insulated Stretchable Conductor for Human Interface.

Soft and stretchable conductors with high electrical conductivity and tissue-like mechanical properties are crucial for both on-skin and implantable electronic devices. Liquid metal-based conductors hold great promise due to their metallic conductivity and minimal stiffness. However, the surface oxidation of liquid metal particles in polymeric matrices poses a challenge in forming a continuous pathway for highly conductive elastic composites. Here, it is reported a printable composite material based on liquid metal and conducting polymer that undergoes a self-assembly process, achieving high conductivity (2089 S cm-1) in the bottom surface while maintaining an insulated top surface, high stretchability (>800%), and a modulus akin to human skin tissue. This material is further applied to fabricate skin-interfaced strain sensors and electromyogram sensors through 3D printing.

The Effects of Transforming Growth Factor-ß1 on the Differentiation of Cell Organoids Composed of Gingiva-Derived Stem Cells.

This study was aimed at evaluating the effects of transforming growth factor-ß on the differentiation and mRNA expression of organoids made out of human mesenchymal stem cells. Cell organoids composed of gingiva-derived stem cells were cultured in the presence of transforming growth factor-ß1 at concentrations ranging from 0, 1, 10, to 20 ng/ml. Evaluations of the cell morphology of the organoids were performed on days 7, 9, 11, and 14. Quantitative cellular viability was completed on day 14. Alkaline phosphatase activity assays were performed to evaluate the differentiation of stem cells on day 14. Real-time polymerase chain reactions were used to determine the expression levels of TGF-ß1, RUNX2, OCN, SOX9, and COL1A1 mRNA on day 14. The stem cells produced well-formed organoids on day 7, and the addition of transforming growth factor-ß1 did not result in relevant changes in their shape. The organoids grew in size and became more intact with longer incubation times. On day 14, the diameters were 222.2 ± 9.6, 186.1 ± 4.8, 197.2 ± 9.6, and 211.1 ± 19.2 m for transforming growth factor-ß1 at final concentrations of 0, 1, 10, and 20 ng/ml, respectively. Quantitative cell viability results from day 14 exhibited no significant difference between the groups (P > 0.05). There was significantly higher alkaline phosphatase activity with the addition of transforming growth factor-ß1 with the highest value for the 1 ng/ml group (P < 0.05). Real-time polymerase chain reaction results demonstrated that the mRNA expression levels of RUNX2, OCN, and SOX were higher in 1 ng/ml but did not reach statistical significance. Treatment with 1 ng/ml of transforming growth factor-ß1 significantly increased COL1A1 mRNA expression at day 14. The application of transforming growth factor-ß1 increased differentiation, which was confirmed by alkaline phosphatase activity and mRNA expression while maintaining cell viability.

Effects of noni on cellular viability and osteogenic differentiation of gingiva-derived stem cells demonstrated by RNA sequencing and quantitative PCR.

Noni fruit (Morinda citrifolia) has been widely used in traditional medicine across tropical and subtropical regions, and is now being paid more attention in Western medicine. The present study aimed to investigate the effects of noni extract on the change in the cellular morphology, maintenance of cellular viability and enhancement of osteogenic differentiation of stem cells. Stem cells obtained from gingiva were cultured where noni extracts existed at concentrations ranging from 10-200 ng/ml. Evaluations of cell morphology and cellular viability were performed. Alkaline phosphatase activity assays were performed to assess the osteogenic differentiation. Alizarin Red S staining was performed to evaluate the calcium deposits in the culture, with the addition of noni extract. Global gene expression was analyzed via next-generation mRNA sequencing. Gene ontology and pathway analyses were performed to determine the associated mechanisms. Validation procedures were performed via quantitative (q)PCR analysis. The addition of noni at concentrations ranging from 10-200 ng/ml did not produce significant morphological changes. There were significantly higher values of cellular viability, with the highest value at 100 ng/ml compared with the control (P<0.05). Furthermore, significantly higher values of alkaline phosphatase activity was noted in the 10 and 100 ng/ml groups compared with the 0 ng/ml group on day 7 (P<0.05). Alizarin Red S staining revealed calcium deposits in each group. In addition, the highest value for Alizarin Red S staining was observed at 100 ng/ml compared with the unloaded control (P<0.05). qPCR analysis demonstrated that the mRNA expression levels of RUNX2, BSP, OCN and COL1A1 increased following treatment with noni. Taken together, the results of the present study suggest that noni extract has enhancing effects on gingiva-derived mesenchymal stem cells, by enhancing cellular viability and osteogenic differentiation.

Factors Affecting Zero-Waste Behaviours of College Students.

This study evaluated the recognition and attitude toward microplastic and zero waste among college students and investigated the factors influencing their zero-waste behaviours. The study was conducted from 20 August 2021 to 10 September 2021, including students at a university in G metropolitan city, Republic of Korea. A total of 196 data were analysed. Statements were developed to verify how the use of disposables and the recognition, attitude, and behaviours related to zero waste were affected during the COVID-19 pandemic. Family type and usage of disposables were the factors affecting zero-waste behaviour in Model 1. In Model 2, which included the subcategory of zero-waste recognition, the health effects of microplastics and environmental preservation were significant factors. In Model 3, which included the subcategory of zero-waste attitude, the health effects of microplastics (ß = 0.149, p = 0.016), use of eco-friendly products (ß = 0.342, p < 0.001), and environmental preservation (ß = 0.317, p < 0.001) were significant factors. The use of plastic products increased dramatically during the COVID-19 pandemic. Research and education are needed to promote zero-waste behaviours with a focus on microplastics. Raising awareness of the health effects of microplastics can enhance the effectiveness of education.

Morphological stability, cellular viability and stem cell marker expression of three-dimensional cultures of stem cells from bone marrow and periodontium.

The aim of the present study was to evaluate the morphology, cellular viability and stem cell marker expression of three-dimensional cultures of bone marrow and gingiva-derived stem cells in different ratios. Stem cell spheroids were made with bone marrow and gingiva-derived stem cells using ratios of 6:0 (Group 1), 4:2 (Group 2), 3:3 (Group 3), 2:4 (Group 4) and 0:6 (Group 5), respectively. The viability of cell spheroids was analyzed using a Live/Dead kit assay and a Cell Counting Kit-8 assay. Total RNA extraction and reverse transcription-quantitative PCR were performed to detect the mRNA expression levels of Nanog and ß-actin in each group. Stem cell spheroids were well formed in silicone elastomer-based concave microwells with different ratios of bone marrow and gingiva-derived stem cells. The shape of the spheroids and their viability were maintained throughout the entirety of the experimental procedure. Statistically significant increases in spheroid diameters were noted in Groups 4 and 5 on day 1 when compared with Group 1 on day 1. There was a significant increase in the cell viability values seen in Group 3 on day 1 when compared with Group 1 on day 1. Highest levels of Nanog expression was seen in Group 3 on day 10, but the increase was not significant when compared with Group 1 on day 1. Co-culturing with higher ratios of gingiva-derived stem cells produced stem cell spheroids with larger diameters and increased cellular viability. This co-culture technique may be used in stem cell therapy with allogenic stem cell transplantation.

Immediate and delayed lateral ridge expansion technique in the atrophic posterior mandibular ridge.

PURPOSE: The lateral ridge expansion technique is used to expand the narrow edentulous ridge for implant placement. The staged approach can be used to split the mandibular ridge to decrease the risk of malfracture during osteotomy. The present study reports the clinical results of a surgical technique that expands a narrow mandibular ridge using an immediate and a delayed lateral expansion technique. MATERIALS AND METHODS: A total of 32 patients with a narrow edentulous posterior mandibular ridge of 2 to 4 mm were included in the present study, and 84 implants were placed. Of the 32 patients, 23 were treated with an immediate lateral expansion technique and 9 with a delayed lateral expansion technique. RESULTS: Of the 23 patients who underwent the immediate lateral expansion technique, a malfracture of the thin buccal cortical plate occurred during ridge splitting in 5 patients. All buccal segments of the 9 patients who underwent the delayed lateral expansion technique fractured as planned at the inferior horizontal corticotomy line favorably. After 4 to 5 months, all implants were stable and surrounded by bone, and ossification of the osteotomy line was obvious. CONCLUSIONS: The lateral ridge expansion technique is effective for horizontal augmentation in the severely atrophic posterior mandibular ridge. The delayed lateral ridge expansion technique can be used more safely and predictably in patients with high bone quality and thick cortex and a narrower ridge in the mandible.

New Pyrimidinone-Fused 1,4-Naphthoquinone Derivatives Inhibit the Growth of Drug Resistant Oral Bacteria.

BACKGROUND: Dental caries is considered to be a preventable disease, and various antimicrobial agents have been developed for the prevention of dental disease. However, many bacteria show resistance to existing agents. METHODS/PRINCIPAL FINDINGS: In this study, four known 1,4-naphthoquinones and newly synthesized 10 pyrimidinone-fused 1,4-naphthoquinones, i.e. KHQ 701, 702, 711, 712, 713, 714, 715, 716, 717 and 718, were evaluated for antimicrobial activity against Enterococcus faecalis, Enterococcus faecium, Staphylococcus aureus, Staphylococcus epidermidis, Streptococcus mutans, Streptococcus sobrinus, Porphyromonas gingivalis, Actinomyces viscosus and Fusobacterium nucleatum. Pyrimidinone-fused 1,4-naphthoquinones were synthesized in good yields through a series of chemical reactions from a commercially available 1,4-dihydroxynaphthoic acid. MIC values of KHQ 711, 712, 713, 714, 715, 716, 717 and 718 were 6.25-50 µg/mL against E. faecalis (CCARM 5511), 6.25-25 µg/mL against E. faecium (KACC11954) and S. aureus (CCARM 3506), 1.56-25 µg/mL against S. epidermidis (KACC 13234), 3.125-100 µg/mL against S. mutans (KACC16833), 1.56-100 µg/mL against S. sobrinus (KCTC5809) and P. gingivalis (KCTC 5352), 3.125-50 µg/mL against A. viscosus (KCTC 9146) and 3.125-12.5 µg/mL against F. nucleatum (KCTC 2640) with a broth microdilution assay. A disk diffusion assay with KHQ derivatives also exhibited strong susceptibility with inhibition zones of 0.96 to 1.2 cm in size against P. gingivalis. Among the 10 compounds evaluated, KHQ 711, 712, 713, 715, 716 and 717 demonstrated strong antimicrobial activities against the 9 types of pathogenic oral bacteria. A pyrimidin-4-one moiety comprising a phenyl group at the C2 position and a benzyl group at the N3 position appears to be essential for physiological activity. CONCLUSION/SIGNIFICANCE: Pyrimidinone-fused 1,4-naphthoquinones synthesized from simple starting compounds and four known 1,4-naphthoquinones were synthesized and showed strong antibacterial activity to the 9 common oral bacteria. These results suggest that these derivatives should be prospective for the treatment of dental diseases caused by oral bacteria, including drug-resistant strains.

Dimethyl Sulfoxide Leads to Decreased Osteogenic Differentiation of Stem Cells Derived from Gingiva via Runx2 and Collagen I Expression.

OBJECTIVES: Dimethyl sulfoxide (DMSO) plays various functions, including cellular functions such as cellular growth. The aim of this study was to evaluate the effects of DMSO on the proliferation and osteogenic differentiation of human gingiva-derived stem cells. MATERIALS AND METHODS: Stem cells derived from gingiva were cultured in the presence of DMSO at concentrations ranging from 0.01 to 10%. STATISTICAL ANALYSIS: We performed a one-way analysis of variance (ANOVA) with post hoc test to determine the differences between the groups using a commercially available program and the level of significance was 0.05. RESULTS: The cells in the control group showed normal fibroblast morphology. The cells treated with 0.01%, 0.01%, 0.1%, and 1% DMSO were morphologically similar to those from the control group on each day. Statistically significant decreases in cell counting kit-8 (CCK-8) values were seen in the 3% and 10% DMSO groups (p < 0.05). A statistically significant decrease in alkaline phosphatase activity was seen in the 3% DMSO group. (p < 0.05). The application of DMSO produced a decrease in alizarin red S staining. The expression of Runx2 and collagen I by immunofluorescence decreased as the dose of lovastatin increased. CONCLUSION: The effects of DMSO on the viability of osteogenic differentiation among stem cells derived from human gingiva were evaluated. Applying DMSO produced decreased cell viability and decreased osteogenic differentiation in this experimental setting. This should be considered when designing and interpreting the data, and a DMSO-free method may be considered for bone regeneration applications.

Bone morphogenetic protein-7 upregulates genes associated with osteoblast differentiation, including collagen I, Sp7 and IBSP in gingiva-derived stem cells.

The present study was performed to evaluate the effects of short-term application of bone morphogenetic protein-7 (BMP-7) on human gingiva-derived mesenchymal stem cells with next-generation sequencing. Human gingiva-derived stem cells were treated with a final concentration of 100 ng/ml BMP-7 and the same concentration of a vehicle control. mRNA sequencing and data analysis were performed along using gene ontology and pathway analysis. RT-qPCR of mRNA of collagen I, Sp7, IBSP and western blot analysis of collagen I, osterix and bone sialoprotein was also performed. A total of 25,737 mRNAs were identified to be differentially expressed. Regarding osteoblast differentiation, 14 mRNAs were upregulated and 10 were downregulated when the results of the BMP-7 at 3 h were compared with the control at 3 h. The expression of collagen I was increased following the application of BMP-7 at 3 h, and this increase was also observed following western blot analysis. The effects of BMP-7 on stem cells were evaluated with mRNA sequencing, and the expression was validated with RT-qPCR and western blot analysis. The short-term application of BMP-7 produced an increased expression of collagen I, which was associated with target genes selected for osteoblast differentiation. This study may provide novel insights into the role of BMP-7 using mRNA sequencing.

A Study of the Effects of Doxorubicin-Containing Liposomes on Osteogenesis of 3D Stem Cell Spheroids Derived from Gingiva.

The objective of the present investigation is to determine the effects of neutral, anionic, and cationic liposomes loaded with doxorubicin with thin-lipid-film-hydration method on the cellular viability and osteogenesis of stem cell spheroids. Spheroid formation and morphology of the three-dimensional spheroid were noted with an inverted microscope. Quantitative cellular viability was assessed using a commercially available kit. Osteogenic potential was evaluated by applying alkaline phosphatase activity and anthraquinone dye of Alizarin Red S. Western blot analysis was performed using collagen I expression. Spheroids were formed in each silicon elastomer-based concave microwell on Day 1. Noticeable changes of the spheroid were seen with a higher concentration of doxorubicin, especially in the cationic liposome group at Days 5 and 7. We found that the application of doxorubicin for 5 days significantly reduced the cellular viability. A higher concentration of doxorubicin produced a significant decrease in alkaline phosphatase activity. Alizarin Red S staining showed that extracellular calcium deposits were evenly noted in each group. An increase of calcium deposits was noted on Day 14 when compared to Day 7. The morphology of the groups with higher concentrations of doxorubicin showed to be more dispersed. We noticed that doxorubicin-loaded cationic liposomes resulted in the highest uptake of the examined cell spheroids and that doxorubicin-loaded liposomes affected the osteogenic differentiation. The implication of this study is that the type of liposome should be selected based on the purpose of the application.

Inhibitory effect of Bacillus velezensis on biofilm formation by Streptococcus mutans.

Streptococcus mutans plays a key role in the development of dental caries and promotes the formation of oral biofilm produced by glucosyltransferases (GTFs). Bacillus velezensis K68 was isolated from traditional fermented foods and inhibits biofilm formation mediated by S. mutans. Gene amplification results demonstrated that B. velezensis K68 contained genes for the biosynthesis of 1-deoxynojirimycin (1-DNJ), a known GTF expression inhibitor. The presence of the GabT1, Yktc1, and GutB1 genes required for 1-DNJ synthesis in B. velezensis K68 was confirmed. Supernatant from B. velezensis K68 culture medium inhibited biofilm formation by 84% when S. mutans was cultured for 48 h, and inhibited it maximally when 1% glucose was added to the S. mutans culture medium as a GTF substrate. In addition, supernatant from B. velezensis K68 medium containing 3 ppb 1-DNJ decreased S. mutans cell surface hydrophobicity by 79.0 ± 0.8% compared with that of untreated control. The supernatant containing 1-DNJ decreased S. mutans adherence by 99.97% and 98.83% under sugar-dependent and sugar-independent conditions, respectively. S. mutans treated with the supernatant exhibited significantly reduced expression of the essential GTF genes gtfB, gtfC, and gtfD compared to that in the untreated group. Thus, B. velezensis inhibits biofilm formation, adhesion, and GTF gene expression of S. mutans through 1-DNJ production.

Evaluation of the secretion and release of vascular endothelial growth factor from two-dimensional culture and three-dimensional cell spheroids formed with stem cells and osteoprecursor cells.

BACKGROUND: Co-culture has been applied in cell therapy, including stem cells, and has been reported to give enhanced functionality. OBJECTIVES: In this study, stem-cell spheroids were formed in concave micromolds at different ratios of stem cells to osteoprecursor cells, and the amount of secretion of vascular endothelial growth factor (VEGF) was evaluated. MATERIAL AND METHODS: Gingiva-derived stem cells and osteoprecursor cells in the amount of 6 × 105 were seeded on a 24-well culture plate or concave micromolds. The ratios of stem cells to osteoprecursor cells included: 0:4 (group 1), 1:3 (group 2), 2:2 (group 3), 3:1 (group 4), and 4:0 (group 5). RESULTS: The morphology of cells in a 2-dimensional culture (groups 1-5) showed a fibroblast-like appearance. The secretion of VEGF increased with the increase in stem cells, and a statistically significant increase was noted in groups 3, 4 and 5 when compared with the media-only group (p < 0.05). Osteoprecursor cells formed spheroids in concave microwells, and no noticeable change in the morphology was noted with the increase in stem cells. Spheroids containing stem cells were positive for the stem-cell markers SSEA-4. The secretion of VEGF from cell spheroids increased with the increase in stem cells. CONCLUSIONS: This study showed that cell spheroids formed with stem cells and osteoprecursor cells with different ratios, using microwells, had paracrine effects on the stem cells. The secretion of VEGF increased with the increase in stem cells. This stem-cell spheroid may be applied for tissue-engineering purposes.

Cellular viability and osteogenic differentiation potential of human gingiva-derived stem cells in 2D culture following treatment with anionic, cationic, and neutral liposomes containing doxorubicin.

The effects of doxorubicin, particularly doxorubicin liposome, on stem cells have remained to be fully elucidated. The aim of the present study was to evaluate the effects of anionic, cationic and neutral liposomes loaded with doxorubicin on the viability and osteogenic differentiation potential of human gingiva-derived stem cells in two-dimensional culture. Doxorubicin-loaded liposomes were prepared using the traditional thin-lipid-film hydration method. Stem cells were seeded on a culture plate and maintained in osteogenic media. The morphology of the stem cells was observed under an inverted microscope. The number of viable cells was determined using a Cell-Counting Kit-8 assay. The alkaline phosphatase activity was assessed and Alizarin Red S staining was performed to evaluate osteogenic differentiation. A higher concentration of doxorubicin caused noticeable changes in the morphology of the stem cells. Decreases in cellular viability were observed after applying doxorubicin. The application of doxorubicin, particularly at higher concentrations, produced a noticeable decrease in alkaline phosphatase activity and Alizarin Red S staining. The present study indicated that application of doxorubicin with or without liposomes reduced the cellular viability and osteogenic differentiation. Among the different treatments, the doxorubicin-loaded cationic liposomes induced the strongest reduction in the cellular viability and osteogenic differentiation in the stem cell culture.

Osteogenic potential of cell spheroids composed of varying ratios of gingiva-derived and bone marrow stem cells using concave microwells.

The aim of the current study was to evaluate cell viability and osteogenic differentiation potential in cell spheroids composed of varying ratios of gingiva-derived and bone marrow stem cells cultured in concave microwells. Cell spheroids were established from bone marrow and gingiva-derived stem cells in ratios of 6:0 (Group 1), 2:1 (Group 2), 3:3 (Group 3), 1:2 (Group 4), and 0:6 (Group 5). On days 3 and 5, the viability of the cell spheroids was qualitatively analyzed using a calcein acetoxymethyl ester working solution and an ethidium homodimer-1 live/dead assay. On days 1, 3, 5 and 7, a quantitative cell viability analysis was performed using a Cell Counting Kit-8. Alkaline phosphatase activity assays were performed using a commercially available kit on day 7 to assess osteogenic differentiation. In addition, reverse transcription-quantitative polymerase chain reaction and western blot analysis were performed to evaluate runt-related transcription factor 2 (Runx2) and osteocalcin expression. The ratio of gingiva-derived to bone marrow stem cells did not affect the stem cell spheroid morphology. No significant changes in cell viability were noted among the different groups following incubation for 7 days. A consistent alkaline phosphatase activity was measured in co-cultured gingiva-derived and bone marrow stem cell spheroids of varying compositions. Runx2 and osteocalcin expression was increased when co-cultured compared with pure gingiva-derived or bone marrow stem cells. In conclusion, stem cell spheroids established by co-culturing maintained morphology, viability and a high osteogenic differentiation potential during the experimental period of 7 days. These spheroids containing human gingiva-derived and bone marrow stem cells may enhance the osteogenic differentiation potential. The use of multicell spheroids may be a simple and effective strategy for improving stem cell therapy.