Cobalt Chloride as a Hypoxia Mimicking Agent Induced HIF-1α and mTOR Expressions of Human Umbilical Cord Mesenchymal Stem Cells

ABSTRACT Objective: To assess the effects of cobalt chloride (CoCl2) as a hypoxia mimicking agent on human umbilical cord mesenchymal stem cells (hUCMSCs) expression of HIF-1α and mTOR for use in regenerative dentistry. Material and Methods: Human umbilical cord mesenchymal stem cells were isolated and then cultured. The characteristics of stemness were screened and confirmed by flow cytometry. The experiment was conducted on hypoxia (H) and normoxia (N) groups. Each group was divided and incubated into 24-, 48-, and 72-hours observations. Hypoxic treatment was performed using 100 µM CoCl2 on 5th passage cells in a conventional incubator (37°C; 5CO2). Then, immunofluorescence of HIF-1α and mTOR was done. Data was analyzed statistically using One-way ANOVA and Tukey's HSD. Results: Significant differences were found between normoxic and hypoxic groups on HIF-1α (p=0.015) and mTOR (p=0.000) expressions. The highest HIF-1α expression was found at 48 hours in the hypoxia group, while for mTOR at 24 hours in the hypoxia group. Conclusion: Hypoxia using cobalt chloride was able to increase human umbilical cord mesenchymal stem cells expression of HIF-1α and mTOR.

Phytochemical, antioxidant, and antibacterial activity of Moringa oleifera nanosuspension against peri-implantitis bacteria: An in vitro study.

Objective: the Moringa oleifera leaf (MO) has active compounds that may be beneficial for peri-implantitis therapy. This research aims to analyze the phytochemical, antioxidant, and antibacterial properties of Moringa oleifera L. nanosuspension (MON) extract in peri-implantitis-related bacteria. Methods: MON extract phytochemical analysis was conducted to examine active compounds such as flavonoids, saponins, quinones, alkaloids, tannins, terpenoids, and steroids. The 2,2-diphenyl-1-picrylhydrazyl (DPPH) assay for antioxidant capacity was evaluated, and gas chromatography-mass spectrometry for the detection of volatile active compounds in MON extract was performed. Turax was used to create MON extract at concentrations of 1% and 2%, and then a particle size analysis was carried out. Prevotella intermedia (Pi), Porphyromonas gingivalis (Pg), Aggregatibacter actinomycetemcomitans (Aa), and Fusobacterium nucleatum (Fn) were tested for antibacterial activity of MON extract, comparing them with doxycycline as the reference drug and using the minimal inhibitory concentration (MIC), minimal bactericidal concentration (MBC), and diffusion zone methods. Results: MON extract has lower antioxidant capacity than vitamin C. Flavonoids, saponins, quinones, alkaloids, tannins, terpenoids, and steroids were found in MON extract. 1% and 2% of MON extract has 10-40 d nm particle size. MIC, MBC and diffusion examination of 1% and 2% MON extract on Aa, Pg, Pi, and Fn were seen at concentrations of 25% and 12.5% with significantly different (p < 0.05) in vitro. Conclusion: MON extract has potential antioxidant activity, and 1% or 2% of MON extract has antibacterial properties toward Aa, Pg, Pi, and Fn at concentrations of 25% and 12.5%, with significant differences.

Human umbilical cord mesenchymal stem cells accelerate and increase implant osseointegration in diabetic rats.

OBJECTIVE: This study was conducted to assess the effect of hUCMSCs injection on the osseointegration of dental implant in diabetic rats via Runt-related Transcription Factor 2 (Runx2), Osterix (Osx), osteoblasts, and Bone Implant Contact (BIC). METHODOLOGY: The research design was a true experimental design using Rattus norvegicus Wistar strain. Rattus norvegicus were injected with streptozotocin to induce experimental diabetes mellitus. The right femur was drilled and loaded with titanium implant. Approximately 1 mm from proximal and distal implant site were injected with hUCMSCs. The control group was given only gelatin solvent injection. After 2 and 4 weeks of observation, the rats were sacrificed for further examination around implant site using immunohistochemistry staining (RUNX2 and Osterix expression), hematoxylin eosin staining, and bone implant contact area. Data analysis was done using ANOVA test. RESULTS: Data indicated a significant difference in Runx2 expression (p<0.001), osteoblasts (p<0.009), BIC value (p<0.000), and Osterix expression (p<0.002). In vivo injection of hUCMSCs successfully increased Runx2, osteoblasts, and BIC value significantly, while decreased Osterix expression, indicating an acceleration of the bone maturation process. CONCLUSION: The results proved hUCMSCs to accelerate and enhance implant osseointegration in diabetic rat models.

Bone Remodeling in Mandible of Wistar Rats with Diabetes Mellitus and Osteoporosis.

OBJECTIVES: This study aimed to determine some of bone molecular expressions and its possible bone remodeling pathway between diabetes mellitus (DM) and osteoporosis model in the mandibular bone of Wistar rats. MATERIALS AND METHODS: Twenty-seven female Wistar rats were divided randomly into control and treatment groups. Treatment groups were injected with streptozotocin intraperitoneally to induce DM (P1) and underwent bilateral ovariectomy to generate osteoporosis (P2). All groups were terminated after 12 weeks. Immunohistochemical and hematoxylin-eosin staining were performed to determine the expression of Runt-related transcription factor 2 (RUNX2), Osterix, vascular endothelial growth factor (VEGF), receptor activator of nuclear factor κB ligand (RANKL), osteoprotegerin (OPG), tartrate-resistant acid phosphatase (TRAP), and observed the osteoblast and osteoclast. Statistical analysis was performed using one-way analysis of variance. RESULTS: The lowest mean of RUNX2 and VEGF expression was found in the P2 group. The lowest mean of Osterix expression was found in the P1 group. Both P1 and P2 groups of osteoblast/osteoclast ratio were decreased. There were no significant differences in the expression of TRAP between all groups; however, increased expression of RANKL/OPG ratio was only found in the P2 group. CONCLUSION: DM and osteoporosis induce changes in the bone remodeling pathway which are represented by a decrease in osteoblast biomarkers and an increase in osteoclast biomarkers.

Anti-Peri-implantitis Bacteria's Ability of Robusta Green Coffee Bean (Coffea Canephora) Ethanol Extract: An In Silico and In Vitro Study.

OBJECTIVE: This study was aimed to investigate RGCBE extract as antioxidant and anti-peri-implantitis bacteria through in vitro study and its potential as antioxidant, antibacterial, anti-inflammatory, antibone resorption, and proosteogenic through in silico study. MATERIALS: AND METHODS: Absorption, distribution, metabolism, excretion and toxicity prediction, molecular docking simulation, and visualization of chlorogenic acid (CGA) and coumaric acid (CA) as anti-inflammatory, antioxidant, and antibacterial were investigated in silico. Inhibition zone by diffusion method, minimum inhibitory concentration (MIC), and minimum bactericidal concentration (MBC) of RGCBE extract against Aggregatibacter actinomycetemcomitans (Aa), Porphyromonas gingivalis (Pg), Fusobacterium nucleatum (Fn), and Prevotella intermedia (Pi) were done. STATISTICAL ANALYSIS: the analysis of variance (ANOVA) difference test, and the post-hoc Tukey's Honest Significant Different (HSD) with a different significance value of p<0.05 RESULTS: GCA and CA compounds are good drug molecules and it has low toxicity. Chlorogenic acid have higher binding activity than coumaric acid to tumor necrosis factor (TNF)-α, nuclear factor (NF)-κB, receptor activation NF-κB (RANK) and its ligand (RANKL), interleukin (IL)-6, IL-10, runt related transcription factor (RUNX2), receptor activator nuclear Kappa beta Ligand-osteoprotegrin osteocalcin (RANKL-OPG), osteocalcin, nuclear factor associated T-cell 1 (NFATc1), tartate resistant acid phosphatase (TRAP), peptidoglycan, flagellin, dectin, Hsp70, and Hsp10 protein. RGCB ethanol extract has high antioxidant ability and it has MIC, MBC, and inhibit the growth of Aa, Pg, Fn, and Pi at 50% concentration with significantly different (p=0.0001 and<0.05). CONCLUSION: RGCB ethanol extract has high antioxidant ability and 50% RGCB ethanol extract may act as strong anti-peri-implantitis bacteria in vitro. In addition, CGA in RGCB potential as antioxidant, antibacterial, anti-inflammatory, antibone resorption, and proosteogenic in silico.

Human umbilical cord mesenchymal stem cells accelerate and increase implant osseointegration in diabetic rats

Abstract Objective This study was conducted to assess the effect of hUCMSCs injection on the osseointegration of dental implant in diabetic rats via Runt-related Transcription Factor 2 (Runx2), Osterix (Osx), osteoblasts, and Bone Implant Contact (BIC). Methodology The research design was a true experimental design using Rattus norvegicus Wistar strain. Rattus norvegicus were injected with streptozotocin to induce experimental diabetes mellitus. The right femur was drilled and loaded with titanium implant. Approximately 1 mm from proximal and distal implant site were injected with hUCMSCs. The control group was given only gelatin solvent injection. After 2 and 4 weeks of observation, the rats were sacrificed for further examination around implant site using immunohistochemistry staining (RUNX2 and Osterix expression), hematoxylin eosin staining, and bone implant contact area. Data analysis was done using ANOVA test. Results Data indicated a significant difference in Runx2 expression (p<0.001), osteoblasts (p<0.009), BIC value (p<0.000), and Osterix expression (p<0.002). In vivo injection of hUCMSCs successfully increased Runx2, osteoblasts, and BIC value significantly, while decreased Osterix expression, indicating an acceleration of the bone maturation process. Conclusion The results proved hUCMSCs to accelerate and enhance implant osseointegration in diabetic rat models.

Human Umbilical Cord Mesenchymal Stem Cell-induced Osterix, Bone Morphogenetic Protein-2, and Tartrate-resistant Acid Phosphatase Expression in Osteoporotic Mandibular Bone.

OBJECTIVES: The aim of this study was to prove that human umbilical cord mesenchymal stem cell (hUCMSC) therapy conducted according to the mandibular osteoporotic model will increase Osterix (Osx) and bone morphogenetic protein-2 (BMP-2) expression, while reducing tartrate-resistant acid phosphatase (TRAP) expression. PKH26 labeling proves that mandibular bone regeneration is produced by hUCMSCs induction. MATERIALS AND METHODS: This study incorporated a true posttest only control group design. Twenty-five female Wistar rats were randomly divided into five groups consisting of the sham surgery (N) group, osteoporotic groups injected with gelatin for 4 weeks (G4) and 8 weeks (G8), and osteoporotic groups injected with hUCMSC-gelatin for 4weeks (SC4) and 8 weeks (SC8). All subjects were provided for BMP-2, Osx, and TRAP on immunohistochemistry examination and PKH-26 labeling. STATISTICAL ANALYSIS: All data were analyzed using ANOVA and Tukey HSD tests with p < 0.05 being considered as statistically significant. RESULTS: Compared with other groups, the highest level of BMP-2 and Osx occurred in the sham surgery (N) and osteoporotic groups injected with hUCMSCs-gelatin (SC), while the lowest level of TRAP was found in SC4. During 4- and 8-week observation periods, the PKH 26 appeared green (fluorescent). CONCLUSIONS: hUCMSC demonstrates high-osteogenic activity and increased osteoporotic mandibular bone regeneration, as shown by increased expression of Osx and BMP-2 and decreased TRAP expression. From the labeling, PKH-26 proved that viable hUCMSCs in gelatin solvent can be present in the mandibular bone and be capable of promoting osteogenic differentiation and increasing mineralization and bone formation in the osteoporotic mandibular bone.

COX-2 induces T cell accumulation and IFN-γ production during the development of chromium allergy.

Chromium (Cr) is commonly added into various metal alloys to improve some mechanical properties such as corrosion resistance, strength, and workability. However, Cr is also known to be a metal allergen for some individuals. Metal allergy is a T cell-mediated disease with symptoms of inflammation and swelling that involve inflammatory cytokines and prostaglandins. Hence, suppressing these inflammation paths by using COX-2 inhibitor might be useful in treating Cr allergy. In this study, mice were used with Cr-induced allergy challenge model. The mice were injected with celecoxib once per day for 7 days one hour after the challenge. Footpad samples were stained with haematoxylin and eosin (H&E), and lymphocytes were isolated from popliteal lymph nodes for the flow cytometric analysis. The results show that both prostaglandin E2 (PGE2), a known mediator of inflammation, and cyclooxygenases (COX)-2 have important roles in the development of Cr allergy. Further, COX-2 inhibitor, celecoxib, was effective in relieving swelling and inflammation in Cr-allergic mice concordant with suppression of IFN-γ production by CD8+ T cells and T cell accumulation in the lymph nodes. Therefore, the inhibition of COX-2 may be a therapeutic target for Cr allergy, and additional molecules in the PGE2 signalling pathway may also be an effective therapeutic target for the treatment of metal allergy.