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 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.

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.

Human Umbilical Cord Mesenchymal Stem Cells Over Platelet Rich Fibrin Scaffold for Mandibular Cartilage Defects Regenerative Medicine

ABSTRACT Objective: To evaluate the regeneration of mandibular cartilage defect after implantation of human umbilical cord mesenchymal stem cells (hUCMSC) over platelet rich fibrin (PRF) as scaffold. Material and Methods: 20 male Wistar rats were randomly divided into four experimental groups consisting of: a control group featuring untreated mandibular defects (C), experimental groups whose mandibular defects were implanted with hUCMSC (E1), mandibular defects implanted with PRF (E2), mandibular defects implanted with hUCMSC and PRF scaffold (E3). The subjects were sacrificed after six weeks of observation for immunohistochemical examination in order to evaluate the expression of Ki67, Sox9, FGF 18, type 2 collagen, and aggrecan, in addition to histology examination to evaluate chondrocyte number and cartilage thickness. Data was analyzed with univariate analysis (ANOVA). Results: The implantation of hUCMSC and PRF scaffold proved capable of regenerating mandibular cartilage defect through the expression of FGF 18, Sox9, Ki67, chondrosis counts, type 2 collagen, aggrecan, and cartilage thickness. The regeneration were significantly higher in group E3. Conclusion: Human umbilical cord mesenchymal stem cells in platelet rich fibrin scaffold proved capable of regenerating mandibular cartilage defect.

Inflammatory and Immunogenic Response of the Tissue after Application of Freeze-Dried Hydroxyapatite Gypsum Puger Scaffold Compared to Freeze-dried Hydroxyapatite Bovine Scaffold.

BACKGROUND: Inflammation is a mechanism or reaction of the natural immune system to defend from external hazards. All foreign objects that enter the body will trigger an immune response in the form of antibodies. In Indonesia, the prevalence of diseases that involve the inflammatory process in the body is high. Freeze-dried hydroxyapatite gypsum puger (HAGP) scaffold is a gypsum powder which is currently under development as a bone replacement material. Freeze-dried hydroxyapatite bovine (HAB) scaffold is a bone substitute material available on the market. OBJECTIVE: To analyze the inflammatory and immunogenic responses in the tissue after application of freeze-dried HAGP scaffold compared to freeze-dried HAB scaffold through mediators of tumor necrosis factor alpha (TNF-α) and immunoglobulin G (IgG) in rats. MATERIALS AND METHODS: This study used Wistar rats. HAGP group and HAB group were applied subcutaneously, settled for 7 and 14 days, then the levels of TNF-α and IgG were measured using enzyme-linked immunosorbent assay. Statistical analysis was done using nonparametric test with the Kruskal-Wallis test. RESULTS: TNF-α levels at day 7 in the HAGP group were nearly equal to the control group, while those in the HAB group were higher. Statistically, the significance was P = 0.184 (P > 0.05). At the 14th day, the level of IgG on the HAGP and HAB groups the level was higher than the control group, statistically it was found P= 0.127. CONCLUSION: freeze-dried HAGP scaffold compared to freeze-dried HAB scaffold did not cause inflammatory and immunogenic response on rats through mediators of TNF-α and IgG.

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.

Human Umbilical Cord Mesenchymal Stem-Cell Therapy to Increase the Density of Osteoporotic Mandibular Bone.

OBJECTIVE: The aim of this study is to evaluate the feasibility of human umbilical cord mesenchymal stem-cell (hUCMSC) therapy in increasing osteoporotic mandibular bone density in a rat model by determining changes in alkaline phosphatase (ALP), osteocalcin, type 1 collagen, and trabecular bone area after treatment. MATERIALS AND METHODS: This research adopted an experimental posttest-only control group design. Thirty female Wistar rats were randomly divided into six groups, namely, a control group with rats postsham surgery (T1), osteoporotic model postovariectomy rats (T2), postovariectomy rats 4 weeks after gelatin injection (T3), postovariectomy rats 8 weeks after gelatin injection (T4), postovariectomy rats 4 weeks after hUCMSC injection (T5), and postovariectomy rats 8 weeks after hUCMSC injection (T6). The rats were all sacrificed for histological and immunohistochemical examinations of ALP, osteocalcin, type 1 collagen, and trabecular bone area. RESULTS: Increased expression of ALP, type 1 collagen, and osteocalcin, as well as increased trabecular bone area, was observed in the treatment groups compared with that in the osteoporotic groups. CONCLUSION: hUCMSCs produce significant osteogenic effects and increase osteoporotic mandibular bone density in the animal model. Increases in bone density are demonstrated by the higher levels of ALP, osteocalcin, and type 1 collagen, as well as increases in the trabecular bone area.

A potential therapy of human umbilical cord mesenchymal stem cells for bone regeneration on osteoporotic mandibular bone.

OBJECTIVE: The aim of this study is to prove that human umbilical cord mesenchymal stem cell (hUCMSC) therapy on mandibular osteoporotic model is able to increase transforming growth factor-beta-1 (TGF)-ß1 expression, Runx2, and osteoblasts. MATERIALS AND METHODS: This research is true experimental posttest control group design. Thirty female Wistar rats were divided into 6 groups randomly, which consisted of sham surgery for control (T1), ovariectomy as osteoporotic group (T2), osteoporotic group injected with gelatine for 4 weeks (T3), 8 weeks (T4) injected with hUCMSC-gelatine for 4 weeks (T5) and 8 weeks (T6). All mice were presented for immunohistochemistry examination for TGF-ß1, Runx2, and histology for osteoblasts. RESULTS: The lowest level of osteoblast was osteoporotic group injected with gelatine in 4 weeks compared to other groups. There were increases of TGF-ß1, Runx2, and osteoblasts from osteoporotic group compared to osteoporotic post-hUCMSC-gelatine injection group. CONCLUSION: The hUCMSC has a high osteogenic effect and increases the osteoporotic mandibular bone regeneration on the animal model that is showed by the increase of the level of TGF-ß1, Runx2, and osteoblasts.