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.

Human umbilical cord mesenchymal stem cells induction in peri-implantitis Rattus norvegicus accelerates and enhances osteogenesis activity and implant osseointegration.

Peri-implantitis additional treatment generally aims to repair damaged tissue through a regenerative approach. Human umbilical cord mesenchymal stem cells (hUCMSCs) produce a high osteogenic effect and are capable of modulating the immune system by suppressing inflammatory response, modulating bone resorption, and inducing endogenous osteogenesis. AIM: This study was intended to discover the effect of hUCMSCs on an implant osseointegration process in peri-implantitis rat subjects as assessed by several markers including interleukin-10 (IL-10), transforming growth factor-ß (TGF-ß), receptor activator of nuclear factor kappa- ß ligand (RANKL), bone morphogenic protein (BMP-2), osterix (Osx), and osteoprotegerin (OPG). MATERIAL AND METHODS: The research design implemented during this study represented a true experimental design incorporating the use of Rattus norvegicus (Wistar strain) as subjects. RESULTS: Data analysed by means of a Brown Forsythe test indicated differences between the increase in BMP-2 expression (p < 0.000) and Osx expression (p < 0.001) and between RANKL expression (p < 0.001, Tukey HSD) and OPG expression (p < 0.000, Games Howell). CONCLUSION: According to the findings of this research, hUCMSCs induction is successful in accelerating and enhancing osteogenic activity and implant osseointegration in peri-implantitis rat subjects.

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.

Effect of Lipopolysaccharide-Induced Apical Periodontitis in Diabetes Mellitus Rats on Periapical Inflammation.

OBJECTIVES: To evaluate periapical inflammation through immunohistochemical analysis of interleukin 6 (IL-6) and tumor necrosis factor α (TNF-a) expression resulting from lipopolysaccharide (LPS)-induced apical periodontitis in diabetes mellitus rats, observed at 14, 28, and 42 days. MATERIALS AND METHODS: Diabetes model on rats was induced by streptozotocin (STZ). Fifteen rats were injected with low-dose STZ for 5 days and waited for 5 days until the blood glucose level was stable and measured above 300 mg/dL confirmed by a digital glucometer. LPS was used to induce apical periodontitis. After performing access cavity, pulpal and root canal extirpation was done on the right mandibular first molar's root canal space of rats, under anesthesia. LPS of 1 mg/mL dose was induced in the pulpal and root canal space. Apical periodontitis was expected 14 days afterward and then, the rats were randomly allocated to three groups. The first group was terminated 14 days after induction and used as control. The second group was observed 28 days after induction, and the third group was observed 42 days after induction. IL-6 and TNF-a expression was analyzed by immunohistochemistry on macrophages in the periapical area. STATISTICAL ANALYSIS: Data were analyzed using one-way ANOVA and continued with the post hoc Tukey HSD test. Significance was considered if p < 0.05. RESULTS: LPS induced apical periodontitis in diabetes mellitus rats at control (14 days), 28 and 42 days observation showed a significant increase in the expression of IL-6 and TNF-a. There were significant differences between the control and observed groups (p < 0.05). The expression of IL-6 in the apical area was not significant at 14 and 28 days (p > 0.05) but increased significantly at 42 days (p < 0.05). The expression of TNF-a in the apical area was significantly increased after 14 days (p < 0.05) and remained stable at 28 and 42 days (p > 0.05). CONCLUSIONS: The periapical inflammation of LPS-induced apical periodontitis in diabetes mellitus rats increased macrophages' expression of IL-6 at 42 days and TNF-a at 28 days.

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.

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.

Calcium Hydroxide Increases Human Umbilical Cord Mesenchymal Stem Cells Expressions of Apoptotic Protease-Activating Factor-1, Caspase-3 and Caspase-9.

PURPOSE: Calcium hydroxide is a gold standard dental material generally used for pulpal and periapical therapy including regenerative endodontic procedures because of its positive properties. However, evaluation about this material on stem cells is limited. Human umbilical cord mesenchymal stem cells (HUCMSCs) are potential to be used in regenerative therapy. Regenerative therapy needs a sustainable cell supply to maintain its regenerative capacity. The aim of this study was to ascertain the apoptosis result of calcium hydroxide on HUCMSCs through the expression of apoptotic protease-activating factor-1 (APAF-1), caspase-3, and caspase-9. MATERIALS AND METHODS: This study used a thawed frozen stock of passage 5 HUCMSCs, grown in minimum essential medium (MEM) alpha containing calcium hydroxide at concentration of 0.1 microgram/mL for 1, 3 and 7 days. Polyclonal antibody with fluorescence isothiocyanate (FITC) label was used to evaluate the expressions. APAF-1, caspase-3, and caspase-9 expressions were recorded and compared on every observation day using fluorescence microscope. Analysis of variance was performed to analyze the significance among the results of treatment groups. The results were concluded significant if p<0.05. RESULTS: The addition of calcium hydroxide in MEM alpha medium increases HUCMSCs expression of APAF-1, caspase-3 and caspase-9 significantly, compared to the control group without calcium hydroxide (p<0.05) in all the times. Day 1 showed the lowest increase followed by higher expressions on day 3 and day 7. CONCLUSION: HUCMSCs express increased APAF-1, caspase-3 and caspase-9 after in-vitro calcium hydroxide exposure. This should be considered when using calcium hydroxide on HUCMSCs for regenerative procedures with regard to other positive properties.

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.

Lipopolysaccharides' Cytotoxicity on Human Umbilical Cord Mesenchymal Stem Cells

Abstract Objective: To show the cytotoxicity of Porphyromonas gingivalis lipopolysaccharide (LPS) on human umbilical cord mesenchymal stem cells (HUCMSCs) to better understand the characteristics for its application in regenerative procedures under periodontopathogen LPS influence. Material and Methods: Ultrapure Porphyromonas gingivalis LPS was used in this study. This research used a frozen stock HUCMSCs, previously confirmed by flow cytometry. The biological characteristics, such as cell morphology, proliferation, and protein expression, were screened. To check the cytotoxicity, HUCMSCs were cultured and divided into two groups, the control group and LPS group with various concentrations from 25 to 0.39 µg/mL. MTT assay was done and the cells were observed and counted. The significance level was set at 5%. Results: The percentage of living HUCMSCs on LPS group were not significantly different among concentrations (p>0.05) from 25 to 0.39 µg/mL, even though there were slight mean decrease between groups, but they were not significant. The duration of 24 hours of exposure of LPS does not significantly lower HUCMSCs viability. Conclusion: LPS does not affect the viability of HUCMSCs. The lower the concentration of LPS, the higher the viability of HUCMSCs.

Cytotoxicity of Calcium Hydroxide on Human Umbilical Cord Mesenchymal Stem Cells

Abstract Objective: To examine the cytotoxicity of calcium hydroxide on human umbilical cord mesenchymal stem cells (HUCMSC) to understand the characteristics for use in regenerative dentistry procedures especially regenerative endodontics. Material and Methods: HUCMSC was isolated, cultured, and confirmed by flow cytometry. The biological characteristics, such as cell morphology, proliferation, and protein expression, were screened. To check the cytotoxicity, HUCMSC was cultured and divided into two groups, the control group (cultured in minimum essential medium (MEM) alpha) and calcium hydroxide group (cultured in MEM alpha and calcium hydroxide). Methyl-thiazole-tetrazolium (MTT) assay was done on different concentrations of calcium hydroxide (0.39 to 25 µg/mL) and the cells were observed and counted. One-way ANOVA test was used with a significance level set at 5%. Results: Flow cytometric analysis confirmed positive of CD73, CD90, CD105, negative of CD45 and CD34. A significant difference was found between the concentration of 6.25 and 3.125 µg/mL (p=0.004). There was no significant difference among 6.25, 12.5 and 25 µg/mL concentrations. There was also no significant difference among 0.39, 0.78, 1.56, and 3.125 µg/mL concentrations. Conclusion: Even though calcium hydroxide is a medicament of choice in clinical endodontics, it decreases the viability of HUCMSC. The lower the concentration of calcium hydroxide, the higher the viability of HUCMSC.

The effect of low-level estrogen in mandibular bone: An in vivo study.

BACKGROUND: Low levels of estrogen can cause osteoporosis and usually occur during a woman's menopausal phase. Osteoporosis can lead to bone resorption, the absence of osseointegration, and implant failure. The aim of this study is to determine the expression of transforming growth factor-beta 1 (TGF-ß1), runt-related transcription factor (RUNX2), and osteoblasts in mandibular rats with low levels of estrogen. MATERIALS AND METHODS: This study is an in vivo experimental research. Female Wistar rats (n = 18) were divided into two groups: (1) Postsham surgery and (2) ovariectomy group. After 12 weeks, the rats were sacrificed to identify the level of estrogen, while histological analysis was conducted to determine the level of osteoblast and the expression of TGF-ß1 and RUNX2. The data were analyzed using t-test (P < 0.05). RESULTS: There were significant lower levels of estrogen and osteoblast among the ovariectomy group compared to the postsham group (P < 0.05). RUNX2 levels were found to be significantly higher in the ovariectomy group than that in the postsham group (P < 0.05). However, there were no significant differences between TGF-ß1 levels within the ovariectomy and postsham groups (P > 0.05). CONCLUSION: Ovariectomy can lead to decreased osteoblastogenesis in mandibular bone by the reduced level of osteoblast and the increased expression of TGF-ß1 and RUNX2.

The Effect of Combination Spirulina-chitosan on Angiogenesis, Osteoclast, and Osteoblast Cells in Socket Models of Hyperglycemic Rattus norvegicus.

BACKGROUND: Prolongation of the inflammatory process in hyperglycemic interferes with bone formation, inhibits the healing process, and triggers bone resorption. A combination of spirulina and chitosan in the tooth socket of Rattus norvegicus is expected to promote the bone remodeling process. This study aimed to evaluate the effect of spirulina and chitosan on angiogenesis, osteoclast, and osteoblast cell in tooth socket models of type 1 diabetes. MATERIALS AND METHODS: A laboratory-based experiment involving 36 R. norvegicus, divided into three groups (nondiabetes mellitus (DM), uncontrolled DM, and controlled DM) and further divided into six subgroups. The controlled groups (K1, K2, and K3) were induced with 3% carboxymethyl cellulose Na, while the treated groups were induced with 12% spirulina and 20% chitosan. On the 14th day, the mandibles of the rats were removed. The capillary lumen, osteoblasts, and osteoclast cells were counted by hypothalamic-pituitary-adrenal examination and the results analyzed by means of Shapiro-Wilk, Levene's, one-way ANOVA, and post hoc Tukey's honestly significant difference test. RESULTS: There was a significant increment in the number of capillary lumen, osteoblast cells, and a decrease in osteoclasts in all three treated groups (P1, P2, and P3). CONCLUSIONS: A combination of spirulina and chitosan can effectively promote the healing process in postextraction sockets of type 1 DM R. norvegicus.