Human gingival mesenchymal stem cells-lyosecretome attenuates adverse effect of hydrogen peroxide-induced oxidative stress on osteoblast cells.

Objective: To determine total protein content, antioxidant activity, and protective ability of lyophilized human gingival mesenchymal stem cells (hGMSCs)-secretome in hydrogen peroxide (H2O2) induced oxidative stress model. Methods: Human GMSCs were cultured to obtain a conditioned medium (secretome), then lyophilized to produce lyosecretome. Total protein was determined by bicinchoninic acid assay (BCA) and SDS-PAGE to improve protein measurements. Antioxidant concentration was measured by ABTS assay, while the protective ability of lyosecretome against oxidative stress was determined by the metabolic activity of osteoblast cells. The study group was divided into a control group (culture medium) and a lyosecretome treatment group (0.0; 0.157, 0.313, 0.625, 1.25, 2.5, 5, and 10 mg/mL + H2O2). Results: Lyosecretome had a protein concentration of 2086.00 ± 0.20 µg/ml, with a molecular weight of 174, 74, 61, 55, and 26 kDa, which are thought to facilitate cell migration, as well as bind cytokines and growth factors. Lyosecretome also provided the highest antioxidant activity of 93.51% at a concentration of 4.8 mg/ml, with an IC50 value of 2.08 mg/ml. The highest cell metabolic activity (79.53 ± 2.41%) was shown in the 1.25 mg/ml lyosecretome treatment group. All concentrations of hGMSC-lyosecretome attenuate the adverse effect of H2O2-induced oxidative stress. Conclusion: Lyosecretome obtained from hGMSCs can maintain metabolic activity in osteoblast cells as protection against H2O2 oxidative stress.

The Effect of Mixed Polymethylmethacrylate and Hydroxyapatite on Viability of Stem Cell from Human Exfoliated Deciduous Teeth and Osteoblast.

OBJECTIVES: Stem cell from human exfoliated deciduous teeth (SHED) has great potential for bone tissue engineering and cell therapy for regenerative medicine. It has been combined with biomaterials such as mixed of polymethylmethacrylate (PMMA) and hydroxyapatite (HA) as candidates for synthetic bone graft biomaterial. The aim of this study was to analyze the toxicity test of mixed PMMA-HA scaffold seeded with SHED and osteoblast in vitro. MATERIALS AND METHODS: SHED was isolated from the pulp of noncarious deciduous teeth and osteoblast cells were cultured, and exposed to PMMA-HA scaffolds with three concentration groups: 20/80, 30/70, and 40/60 for 24 hours. Cytotoxicity test was performed by MTT assay to cell viability. STATISTICAL ANALYSIS: Data were analyzed using IBM SPSS Statistics 25, one-way analysis of variance followed by least significant difference test, considering the level of significance p-value less than 0.05 RESULTS: The percentage of SHED's viability was best in the PMMA-HA group with concentrations of 20/80, followed by 30/70, and 40/60 with 87.03, 75.33, and 65.79%, respectively. The percentage of osteoblast cell's viability was best in the PMMA-HA group with concentrations of 20/80, followed by 30/70, and 40/60 with 123.6, 108.36, and 93.48%, respectively. CONCLUSIONS: Mixed PMMA-HA was not toxic for the SHED and osteoblast. This characteristic is the initial requirement to be proposed as an alternative material for healing alveolar bone defects. In vivo animal research is mandatory to confirm the use of PMMA-HA on the alveolar defect model.

RUNX2 and ALP expression in osteoblast cells exposed by PMMA-HAp combination: An in vitro study.

Objective: To observe the expression of Runt-Related Transcription Factors 2 (RUNX2) and Alkaline Phosphatase (ALP) markers in osteoblast cell cultures exposed to Polymethylmethacrylate (PMMA) combined with hydroxyapatite (HAp) material to improve osteointegration of bone implants. Methods: Sample of PMMA and HAp materials with a mixture of PMMA with HAp made from limestone as natural source which processed through Balai Besar Keramik (HApBBK) in the first group and a mixture of PMMA with HAp made from bovine bone which processed through Good Manufacturing Practice (HApGMP) in the second group. Twenty-four fetal rat calvarie osteoblast cell cultures were randomly divided into 6 groups: 7- and 14-day control group, 7 and 14 days PMMA-HApGMP group, 7 and 14 days PMMA-HApBBK group. The expression of RUNX2 and ALP was seen by immunocytochemical examination. Result: The one-way ANOVA with a significance value of 0.000 (p < 0.05). There was an increase in RUNX2 and ALP expressions on both PMMA-HApBBK and PMMA-HApGMP groups on days 7 and 14 in osteoblast cell cultures. Conclusion: The PMMA-HApBBK and PMMA-HApGMP showed an increase in the RUNX2 and ALP expression in osteoblast cell cultures which indicates a potential increase of osseointegration of bone implants.

Expression of VEGF and BMP-2 in Osteoblast cells exposed to a combination of polymethylmethacrylate (PMMA) and hydroxyapatite (HAp).

Objectives: Polymethylmethacrylate (PMMA) has been widely used, but it has several fallback properties in its interaction with bone tissue, so the addition of hydroxyapatite (HAp) material aims to improve the biocompatibility, regeneration process, and osteointegration of bone implants. The HAp material can be sourced from bovine bone and processed through Good Manufacturing Practice from Tissue Bank (HApGMP), and from limestone (CaCO3) processed by Balai Besar Keramik (HApBBK).This study was to observe the expression of vascular endothelial growth factor (VEGF) and Bone morphogenetic protein-2 (BMP2) in cultured osteoblasts exposed to PMMA-HApGMP and PMMA-HApBBK as implant candidate materials. Methods: Sample of PMMA and HAp materials with a mixture of PMMA and HApBBK in the first group and a mixture of PMMA and HApGMP in the second group. Twenty-four fetal rat calvarie osteoblast cell cultures were randomly divided into 6 groups: 7- and 14-day control group, 7 and 14 days PMMA-HApGMP group, 7 and 14 days PMMA-HApBBK group. The expression of VEGF and BMP-2 was seen by immunocytochemical examination. Results: The one-way ANOVA with a significance value of 0.000 (p < 0.05). BMP-2 and VEGF expression was increased in the 7- and 14-days groups after exposure to PMMA-HApGMP and PMMA-HApBBK. Conclusion: The application of PMMA-HApGMP and PMMA-HApBBK showed an increase in the expression of VEGF and BMP-2 in osteoblast cell cultures which indicates a potential increase in the accelerated angiogenesis and osteogenesis in the bone regeneration process of bone implants.

Study of Alveolar Bone Remodeling Using Deciduous Tooth Stem Cells and Hydroxyapatite by Vascular Endothelial Growth Factor Enhancement and Inhibition of Matrix Metalloproteinase-8 Expression in vivo.

Background: Periodontitis progression is characterized by alveolar bone loss, and its prevention is a major clinical problem in periodontal disease management. Matrix metalloproteinase-8 (MMP-8) has been shown to adequately monitor the treatment of chronic periodontitis patients as gingival crevicular fluid MMP-8s were positively associated with the severity of periodontal disease. Moreover, modulating the vascular endothelial growth factor (VEGF) levels in bones could be a good way to improve bone regeneration and cure periodontitis as VEGF promotes endothelial cell proliferation, proteolytic enzyme release, chemotaxis, and migration; all of which are required for angiogenesis. Purpose: The aim of this study was to determine the effect of hydroxyapatite incorporated with stem cells from exfoliated deciduous teeth (SHED) in Wistar rats' initial alveolar bone remodeling based on the findings of MMP-8 and VEGF expressions. Methods: A hydroxyapatite scaffold (HAS) in conjunction with SHED was transplanted into animal models with alveolar mandibular defects. A total of 10 Wistar rats (Rattus norvegicus) were divided into two groups: HAS and HAS + SHED. Immunohistochemistry staining was performed after 7 days to facilitate the examination of MMP-8 and VEGF expressions. Results: The independent t-test found significant downregulation of MMP-8 and upregulation VEGF expressions in groups transplanted with HAS in conjunction with SHED compared with the HAS group (p < 0.05). Conclusion: The combination of SHED with HAS on alveolar bone defects may contribute to initial alveolar bone remodeling as evident through the assessments of MMP-8 and VEGF expressions.

The differences in the number of fibroblasts and blood vessels after the topical and systemic administration of Lactobacillus casei Shirota probiotics for the treatment of traumatic ulcers in Wistar rats (Rattus norvegicus).

BACKGROUND AND AIM: The use of drugs as a therapy for traumatic ulcers may lead to drug resistance and other side effects. Lactobacillus casei Shirota can affect the number of fibroblasts and blood vessels in wound healing. The aim of this study was to investigate the difference in the number of fibroblast cells and blood vessels after the topical and systemic administration of L. casei Shirota probiotics in Wistar rats with traumatic ulcer. MATERIALS AND METHODS: Overall, 36 healthy male Wistar rats aged 2-3 months old and weighing 175-250 g in body weight were used as a sample. Traumatic ulcer was made on the labial fornix incisive inferior. The subject rats were divided into groups: (1) A control group over 3 days, (2) a group that used distilled water over 7 days, (3) a group that underwent topical treatment over 3 days, (4) a group that used probiotics administered topically over 7 days, (5) a group that underwent systemic treatment over 3 days, and (6) a group that took oral probiotics for the traumatic ulcers over 7 days. The number of fibroblasts and blood vessels was observed through a hematoxylin-eosin examination. RESULTS: Based on the results of the study, a significant difference was observed in the number of fibroblasts (p=0.00) and blood vessels (p=0.018) in the 3-day topical group that underwent a 3-day systemic administration of probiotics compared with the number of fibroblast cells in the 7-day topical group and 7-day systemic group (p=0.00). CONCLUSION: Overall, significant differences were observed in the number of fibroblasts and blood vessels in Wistar rats with traumatic ulcer after undergoing the topical and systemic administration of L. casei Shirota probiotics.

Obesity correlated to a higher risk of acquiring periodontitis: a cross-sectional study.

BACKGROUND: The present study aimed to investigate the correlation between obesity and periodontitis, among other risk factors for periodontitis.   Methods:  In total, 262 Indonesian male and female subjects were analysed for body mass index (BMI), oral hygiene, plaque index, and clinically evaluated periodontitis. Statistical analysis was performed using Spearman tests and Pearson chi-square tests to estimate the correlation between BMI and periodontitis. Multivariate binary logistic analysis was conducted between covariate and periodontitis. P<0.05 was considered as statistically significant.   Results: The prevalence of obesity was 48.47%. There were positive correlations between BMI and periodontal status for healthy-mild periodontitis, moderate, and severe periodontitis respectively. BMI and periodontitis crude odds ratio (OR) = 2.31 (95% CI 1.41-3.78); p < 0.05, adjusted OR of BMI among other variables, was 1.88 (95%CI 1.05-3.37); p < 0.05. Exploration of the ROC curve found a BMI cut off point of 24.785 kg/m2.  Conclusion: Obesity by BMI measurement of ≥ 25kg/m2 correlated to a higher risk of acquiring periodontitis compared to normal-weight individuals.

Immunohistochemical analysis of stem cells from human exfoliated deciduous teeth seeded in carbonate apatite scaffold for the alveolar bone defect in Wistar rats ( Rattus novergicus).

Background: Stem cells from human exfoliated deciduous teeth (SHED) seeded in carbonate apatite scaffold (CAS) may have multiple functions that could be used to regenerate the alveolar bone defects. The purpose of this study is to examine the ability of SHED and CAS in alveolar bone defects using an immunohistochemical analysis. Methods: ten three-month-old healthy male Wistar rats (R. novergicus) that weighed between 150-250 grams (g) were used as animal models. A simple blind random sampling method was used to select the sample that was assigned to the study group for CAS and SHED seeded in CAS (n=5). The animal study model of the alveolar bone was established by extracting the anterior mandible teeth. Rodent anesthesia was applied to relieve the pain during the procedure for all test animals. Immunohistochemistry was performed after seven days to facilitate the examination of the receptor activator of NF-κß ligand (RANKL), osteoprotegrin (OPG), transforming growth factor-ß (TGF-ß), vascular endothelial growth factor (VEGF), runt-related transcription factor 2 (RUNX2), alkaline phosphatase (ALP), osteocalcin, and osteopontin expression. The data was analyzed using the unpaired t-test (p<0.01) and Pearson's correlation test (p<0.05). Results: The OPG, RUNX2, TGF-ß, VEGF, ALP, osteocalcin, and ostepontin expressions were higher in SHED seeded in CAS than CAS only with a significant difference between the groups (p<0.01). Furthermore, the RANKL expression was lower in SHED seeded in CAS compared to CAS only. There was a strong reverse significant correlation between OPG and RANKL expression (p<0.05). Conclusions: The number of osteogenic marker expressing cells, such as OPG, RUNX2, TGF-ß, VEGF, ALP, osteocalcin, and ostepontin, increased. However, RANKL expression in the alveolar bone defects that were implanted with SHED seeded in CAS did not increase after seven days.

Exfoliated Human Deciduous Tooth Stem Cells Incorporating Carbonate Apatite Scaffold Enhance BMP-2, BMP-7 and Attenuate MMP-8 Expression During Initial Alveolar Bone Remodeling in Wistar Rats (Rattus norvegicus).

BACKGROUND: Post-tooth extraction socket preservation is necessary due to alveolar bone resorptive patterns through regenerative dentistry approaches that involve the use of stem cells, scaffold and growth factor. Stem cells derived from human exfoliated deciduous teeth (SHED) are known to potentially possess the osteogenic ability. Meanwhile, carbonate apatite scaffold (CAS) can act as a biocompatible scaffold capable of supporting mesenchymal stem cells (MSCs) to proliferate and differentiate optimally. The aim of this study is to investigate the expression of bone morphogenic protein-2 and 7 (BMP2, BMP7) and Matrix Metalloproteinase-8 (MMP-8) after the transplantation of SHED-incorporated CAS during in vivo bone remodeling. MATERIAL AND METHODS: A total of 14 healthy, male, Wistar rats, whose mandible anterior teeth were extracted by means of sterile needle holder clamps, constituted the subjects of this study of alveolar bone defects. Two research groups were created: a control group (CAS) as group I and an experimental group (CAS + SHED) as group II. SHED with a density of 106 cells were incorporated into CAS before being transplanted into the experimental group. After 7 days, all the animals were sacrificed and their mandible anterior region extracted. The BMP2, BMP7 and MMP-8 expression were subsequently analyzed by means of immunostaining. An unpaired t-test was conducted to analyze the treatment and control group (p<0.01) data. RESULTS: The expression of BMP-2 and BMP-7 was higher in group II compared to group I. Meanwhile, the level of MMP-8 was lower in group II than group I. There was greater significant increased expression of BMP-2 and BMP-7 expression in Group II compared to Group I. There was significant decreased expression of MMP-8 between group II than group I (p<0.01). CONCLUSION: SHED-incorporated CAS can enhance BMP-2 and BMP-7 expression while attenuating MMP-8 expression during in vivo alveolar bone remodeling.

Combined Hydroxyapatite Scaffold and Stem Cell from Human Exfoliated Deciduous Teeth Modulating Alveolar Bone Regeneration via Regulating Receptor Activator of Nuclear Factor-Κb and Osteoprotegerin System.

BACKGROUND: Tissue engineering using Stem cell from Human Exfoliated Deciduous Teeth (SHED) and a natural biomaterials biomaterial scaffold has become a promising therapy for the alveolar bone defect. The aim of this study was to analyze the Osteoprotegerin (OPG) and Receptor Activator of NF-Κb ligand (RANKL) expression after the application of Hydroxyapatite scaffold and SHED. METHODS: A laboratory experimental research with a post test-only control group. 14 male Wistar rats weighing from 260 to 280 g were used as the animal study. The animals were randomly assigned to an experimental group Hydroxyapatite scaffold (group I) and Hydroxyapatite scaffold combined with SHED (group II). The alveolar bone defect in the animal study model was affected by extracting anterior mandible teeth. Immunostaining was performed after 8 weeks in order to facilitate the examination of OPG and RANKL expression. Data were analyzed by independent t test. The correlation between OPG and RANKL expression were analyzed using Pearson's correlation test (P<0.05). Statistical analysis was performed using R statistical software version 3.4.0. RESULTS: The independent t test showed that the differences were statistically significant. OPG expression in Group I (6.0±1.00) was lower than in Group II (11.6±1.14) (P=0.0004). The independent t test showed that the differences were statistically significant. RANKL expression for Group I (12.67±2.08) and Group II (4.80±1.304) showed a statistically significant difference (P=0.0005). CONCLUSION: Hydroxyapatite scaffold and SHED increase Osteoprotegerin and decrease Receptor Activator of NF-κB Ligand expression with high potential as an effective agent in alveolar bone defect regeneration.

Regeneration of Salivary Gland Defects of Diabetic Wistar Rats Post Human Dental Pulp Stem Cells Intraglandular Transplantation on Acinar Cell Vacuolization and Interleukin-10 Serum Level

Abstract Objective: To investigate the regeneration of rat's salivary gland diabetic defect after intraglandular transplantation of Human Dental Pulp Stem Cells (HDPSCs) on acinar cell vacuolization and Interleukin-10 (IL-10). Material and Methods: HDPSCs isolated from the dental pulp of first premolars #34. HDPSCs from the 3rd passage was characterized by immunocytochemistry of CD73, CD90, CD105 and CD45. Twenty-four male Wistar rats, 3-month-old, 250-300 grams induced with Streptozotocin 30 mg/kg body weight to create diabetes mellitus (DM) divided into 4 groups (n=6); positive control group on Day-7; positive control group on Day-14; treatment group Day-7 (DM+5.105HDPSCs); treatment group on Day-14. On Day-7 and Day-14, rats were sacrificed. Histopathological examination performed to analyze acinar cells vacuolization while Enzyme-linked Immunoabsorbent Assay to measure IL-10 serum level. Data obtained were analyzed statistically using multiple comparisons Bonferroni test, Kruskal Wallis, Shapiro-Wilk and Levene's test result Results: The highest acinar cell vacuolization found in control group Day 14 (0.239 ± 0.132), meanwhile the lowest acinar cell vacuolization found in treatment group Day 7 (0.019 ± 0.035) with significant difference (p=0.003). The highest IL-10 serum level found in treatment group Day 14 (175.583 ± 120.075) with significant difference (p=0.001) Conclusion: Transplantation of HDPSC was able to regenerate submandibular salivary gland defects in diabetic rats by decreasing acinar cell vacuolization and slightly increase IL-10 serum level.

Viability test of fish scale collagen (Oshpronemus gouramy) on baby hamster kidney fibroblasts-21 fibroblast cell culture.

AIM: This study aims to examine the toxicity of collagen extracted from gouramy fish scales (Oshpronemus gouramy) by evaluating its viability against baby hamster kidney fibroblasts-21. MATERIALS AND METHODS: Collagen was extracted from gouramy fish scales (O. gouramy) with 6% acetic acid. Its results were analyzed using Fourier-transform infrared spectroscopy and freeze-dried technique. Its morphology then was analyzed with scanning electron microscope. Afterward, 3-(4.5-dimethylthiazole-2-yl)2.5-diphenyl tetrazolium bromide assay was conducted to compare cells with and without fish scale collagen treatment. RESULTS: Collagen extracted from gouramy fish scales had no influence statistically on cultured fibroblast cells with a statistical significance (2-tailed) value of0.754 (p>00025). CONCLUSION: Collagen extracted from gouramy fish scales has high viability against BHK21 fibroblast cells.

Transforming Growth Factor-ß1 and Runt-related Transcription Factor 2 as Markers of Osteogenesis in Stem Cells from Human Exfoliated Deciduous Teeth Enriched Bone Grafting.

BACKGROUND: Stem cells from human exfoliated deciduous teeth (SHED) are one source of adult stem cells which can proliferate and differentiate into many types of tissues than any other stem cells. SHED represent potential stem cells for therapeutic therapy and tissue engineering. AIMS: The aim of this study was to compare the expression of transforming growth factor-ß1 (TGF-ß1) and runt-related transcription factor 2 (RUNX2) in hydroxyapatite (HA) scaffold with SHED. SUBJECTS AND METHODS: Eight experimental animals were divided into two groups. The first group was transplanted with HA and the second with HA and SHED. The expression of TGF-ß1 and RUNX2 was seen 21 days later by means of immunohistochemical analysis. STATISTICAL ANALYSIS USED: Data were analyzed using an independent t-test with a significance level of 5%. RESULTS: The analysis results of an independent t-test showed a significant difference between the two groups. The second group given HA with SHED showed a significantly higher expression of TGF-ß1 and RUNX2 than that of the first group. CONCLUSIONS: Expression of TGF-ß1 and RUNX2 occurs after the application of HA with SHED, while TGF-ß1 and RUNX2 expression in the HA with SHED group was significantly higher than in the group without SHED.