On the last day of the wound healing experiment, the animals were anesthetized using Ketamine HCl (50 mg/kg, i.p.), euthanized, and specimens of wound tissue with the adjacent healthy tissue were collected. The collected samples were fixed in 10% formalin and were subjected to routine histopathological tissue examination. The wound tissue specimen was sectioned with a microtome (Leica RM 2245) and then stained with hematoxylin-eosin. The prepared tissue slide was examined under a light microscope. To evaluate the collagen content, the wound tissue specimen was sectioned using a microtome, stained with Van Gieson stain for collagen fiber, and examined under a microscope (Leica DM IRM, Leica Microsystems, Wetzlar, Germany).
Leica dm irm
Manufactured by Leica Microsystems
Sourced in Germany
The Leica DM IRM is an inverted research microscope designed for advanced microscopy applications. It features a modular construction, allowing for customization to suit various experimental needs. The core function of the Leica DM IRM is to provide high-quality optical performance and versatility in a laboratory setting.
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Lab products found in correlation
Fetal bovine serum (fbs), by Thermo Fisher Scientific (5 mentions)
Streptomycin, by Merck Group (3 mentions)
L glutamine, by Merck Group (3 mentions)
Rm2245, by Leica camera (2 mentions)
Ascorbic acid 2 phosphate, by Merck Group (2 mentions)
Listerine zero, by Johnson & Johnson (1 mentions)
α mem, by Thermo Fisher Scientific (1 mentions)
Microplate reader, by Agilent Technologies (1 mentions)
α minimal essential medium, by Thermo Fisher Scientific (1 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (1 mentions)
Penicillin, by Merck Group (1 mentions)
Mc3t3 e1 cell, by American Type Culture Collection (1 mentions)
Cck 8, by Dojindo Laboratories (1 mentions)
13 protocols using leica dm irm
1
Histopathological Analysis of Wound Healing
2
Evaluation of Antimicrobial Mouthwash Effects
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Figure 1 shows the overview of the study design. Six mouthwashes were tested in this study: (1) benzydamine hydrochloride (150 mg/100 mL; Tantum, Sama Pharm Co. Ltd., Wonju-si, Gangwon-do, Korea); (2) cetylpyridinium chloride (50 mg/100 mL) and benzalkonium chloride ( GUM, Sunstar Inc., Osaka, Japan); (3) methyl salicylate, L-menthol, eucalyptol, and thymol (IP, 3M, St. Paul, MN, USA); (4) sodium fluoride (0.2 mg/1 mL) , xylitol, and chitosan (Cool Spearmint, 3M); and (5) sodium fluoride, xylitol, and chitosan (Mild Muscat, 3M). The treatment times were 30 seconds, 90 seconds, and 270 seconds. An untreated culture sample served as the control. The morphological changes were observed under an inverted microscope (Leica DM IRM, Leica Microsystems, Wetzlar, Germany) after each treatment.
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3
Fabrication and Evaluation of BMSC Spheres
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Ethical approval was granted regarding the present study through the Institutional Review Board of Seoul St Mary's Hospital, College of Medicine, the Catholic University of Korea (number: KC18SESI0083), and all experiments were carried out following the relevant guidelines. Human bone marrow mesenchymal stem cells (BMSCs, Catholic MASTER cells) were obtained from the Catholic Institute of Cell Therapy (CIC, Seoul, Korea). Isolation and propagation of the BMSCs were performed following previously reported methods (12 (link)). CIC verified that all samples showed >90% positive CD 73 and CD 90 expression. We seeded the cells on a culture dish. We removed the cells that were not attached to the dish. We refreshed the culture medium every 2 or 3 days, and grew the cells in the incubator with 95% O2 and 5% CO2 at 37˚C.
Fig. 1 shows an overview of the study's design. We used commercially available concave microwells (H389600, StemFIT 3D; MicroFIT) to fabricate stem cell spheres. We loaded a total of 1x106 cells in each well and evaluated the cell response. We treated cell spheres made of bone marrow mesenchymal stem cells with BMP-2 at predetermined concentrations of 0, 10 and 100 ng/ml. We evaluated the morphological characteristics using an inverted microscope (Leica DM IRM, Leica Microsystems). Morphological evaluation of the spheres was conducted on days 1, 3, 7, 14, and 21.
4
Stem-Cell Spheroid Formation Optimization
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Stem-cell spheroids were formed in silicon elastomer-based concave microwells (StemFIT 3D; MicroFIT, Seonnam, Republic of Korea) with 600 µm diameters. Subsequently, 4×105 (group A) or 8×105 (group B) stem cells were seeded in each concave micromold and cultured to investigate cellular behavior (Fig. 1 ). The difference between group A and group B was only the number of cells per spheroid. Cell aggregation and spheroid formation were observed and images were captured using an inverted microscope (Leica DM IRM; Leica Microsystems GmbH, Wetzlar, Germany). The diameters of spheroids were measured from the captured images.
5
Mouthwash Efficacy on Oral Cell Morphology
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Figure 1 shows the overview of the study design. Six mouthwashes were applied for this study: (1) a 0.12% chlorhexidine digluconate solution (CHX, Hexamedine, Bukwang, Seoul, Korea); (2) a solution containing essential oils (LIS Citrus, Listerine® Citrus, Johnson & Johnson, Bangkok, Thailand); (3) a solution containing essential oils without alcohol (LIS Zero, Listerine® Zero, Johnson & Johnson); (4) Garglin® Regular containing CPC (GGN, Dong-A Pharmaceutical Co., Seoul, Korea); (5) Garglin® Medical containing essential oils (GGN Med, Dong-A Pharmaceutical Co.); and (6) Garglin® Child containing sodium fluoride (GGN Child, Dong-A Pharmaceutical Co.). Each well was treated with one of the six mouthwashes for 30 sec, 1 min and 30 sec (1.5 min), or 4 min and 30 sec (4.5 min). An untreated culture well served as a control. The morphological changes were observed under an inverted microscope (Leica DM IRM, Leica Microsystems, Wetzlar, Germany) after each treatment.
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6
Osteogenic Differentiation of Stem Cells
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A total of 4.5x105 cells were grown in each well with osteogenic media comprising α-MEM (Gibco; Thermo Fisher Scientific, Inc.), 38 µg/ml dexamethasone, 2 mg/ml glycerophosphate disodium salt hydrate, 10 mM ascorbic acid 2-phosphate and 200 mM L-glutamine on days 3, 7, 10 and 14. Alkaline phosphatase activity was evaluated using a commercially available assay kit (cat. no. K412-500; BioVision, Inc.). The absorbance was measured at 405 nm after mixing a 5 mM p-nitrophenylphosphate substrate with cell lysates using assay buffer (cat. no. K412; BioVision, Inc.) and incubating it at 25˚C for 40 min. Comparisons were made between the groups, as the same number of cells was loaded in each group. The assays were performed three times. Stem cell spheroids were stained with 2% Alizarin Red S at room temperature for 30 min after fixing the cell spheroids with 4% paraformaldehyde at room temperature for 15 min and washing them with deionized water twice on day 14(18 (link)). The degree of osteogenesis was evaluated by measuring the relative intensity of Alizarin red S staining using an inverted light microscope at x100 magnification (Leica DM IRM; Leica Microsystems GmbH).
7
Osteogenic Differentiation Assay
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The cells (within 10 passages) were seeded in 96-well plates at a density of 2.0×103 cells/well, and the cells were cultured in osteogenic medium. The osteogenic medium consisted of α-minimal essential medium (Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA) containing 15% fetal bovine serum (Gibco; Thermo Fisher Scientific, Inc.), 100 U/ml penicillin, 100 µg/ml streptomycin (Sigma-Aldrich; Merck KGaA), 200 mM L-glutamine (Sigma-Aldrich; Merck KGaA), 10 mM ascorbic acid 2-phosphate (Sigma-Aldrich; Merck KGaA), 38 µg/ml dexamethasone and 2 mg/ml glycerophosphate disodium salt hydrate. The morphology of the cells was observed under an inverted microscope (Leica DM IRM, Leica Microsystems, Wetzlar, Germany) on days 1, 3 and 7.
On days 1, 3 and 7, a cell viability analysis of the stem cell spheroids cultured in osteogenic medium was performed. Cell Counting Kit-8 (CCK-8) stain (Dojindo, Kumamoto, Japan) was added to the cultures and the stem cells were incubated for 1 h at 37°C. Viable cells were identified by CCK-8 staining, which relies on the ability of mitochondrial dehydrogenases to oxidize the dye into a colored formazan product. A microplate reader (BioTek, Winooski, VT, USA) was used to measure the spectrophotometric absorbance of the samples at 450 nm.
On days 1, 3 and 7, a cell viability analysis of the stem cell spheroids cultured in osteogenic medium was performed. Cell Counting Kit-8 (CCK-8) stain (Dojindo, Kumamoto, Japan) was added to the cultures and the stem cells were incubated for 1 h at 37°C. Viable cells were identified by CCK-8 staining, which relies on the ability of mitochondrial dehydrogenases to oxidize the dye into a colored formazan product. A microplate reader (BioTek, Winooski, VT, USA) was used to measure the spectrophotometric absorbance of the samples at 450 nm.
8
Tacrolimus Effects on Cell Morphology
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The cells were plated at a density of 2.0×103 cells/well in 96-well plates. The cells were incubated in α-MEM containing 15% fetal bovine serum (Gibco; Thermo Fisher Scientific Inc., Waltham, MA, USA), 100 U/ml penicillin and 100 µg/ml streptomycin in the presence of tacrolimus at final concentrations ranging of 0 (control), 0.001, 0.01, 0.1, 1, 10 and 100 µg/ml. Tacrolimus was dissolved in dimethyl sulfoxide (DMSO; Sigma-Aldrich) and filter-sterilized. Equal quantities of DMSO were added to each culture sample to offset the influence of this dissolving vehicle. The morphology of the cells was viewed under an inverted microscope (Leica DM IRM, Leica Microsystems, Wetzlar, Germany) on days 1, 3, 5 and 7. The images were saved as JPEGs.
9
Stem Cell Spheroid Formation in Microwell Culture
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Stem cell spheroids were formed in the silicon elastomer-based concave microwells (StemFIT 3D; MicroFIT, Seongnam, Korea) 600 µm in diameter. A total of 6×105 gingiva-derived stem cells and murine osteoprecursor cells (MC3T3-E1 cells; American Type Culture Collection, Manassas, VA, USA) at different ratios were seeded into the micromolds and subsequently cultured at 37°C in α-minimum essential medium (α-MEM) containing 15% fetal bovine serum (Gibco; Thermo Fisher Scientific, Inc.), 100 U/ml penicillin, 100 µg/ml streptomycin, 200 mM L-glutamine and 10 mM ascorbic acid 2-phosphate (all Sigma-Aldrich; Merk KGaA) to investigate cellular behavior at days 1, 3, 5, and 7. The ratios between gingiva-derived stem cells and osteoprecursor cells were as follows: 0:6 (group 1); 2:4 (group 2); 3:3 (group 3); 4:2 (group 4); and 6:0 (group 5; Fig. 1 ). Cell aggregation and cell spheroid formation were observed and images were captured using an inverted microscope (Leica DM IRM; Leica Microsystems GmbH, Wetzlar, Germany).
10
Alizarin Red S Staining for Mineralization
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Alizarin Red S staining was performed on days 7 and 14. In brief, the cells were washed twice with PBS, fixed with 4% paraformaldehyde at room temperature and rinsed twice with deionized water. The cultures were then stained with Alizarin Red S for 30 min at room temperature. To remove non-specifically bound dye, the cultures were washed three times with deionized water. The morphology was evaluated using an inverted microscope (Leica DM IRM; Leica Microsystems).
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