Polymethylmethacrylate-hydroxyapatite antibacterial and antifungal activity against oral bacteria: An in vitro study.

Objective: Reconstruction of alveolar bone defects resulting from aging, trauma, ablative surgery or pathology, remains a significant clinical challenge. The objective of this study was to investigate the antibacterial and antifungal activities of mixed polymethylmethacrylate-hydroxyapatite (PMMA-HA) against oral microorganisms. Our findings could provide valuable insights into the prospective application of PMMA-HA as a synthetic bone graft material to manage alveolar bone defects via tissue engineering. Methods: HA powder was obtained from the Center for Ceramics in Indonesia and PMMA granules were obtained from HiMedia Laboratories; these were prepared in 20:80, 30:70, and 40:60 ratios. The antibacterial diffusion method was then performed against Staphylococcusaureus, Aggregatibacter actinomycetemcomitans, Porphyromonas gingivalis, and Fusobacterium nucleatum, while the antifungal diffusion method was used to test against Candida albicans. Standardized protocols were used for microbial culturing and inhibition zones were measured with digital calipers. Statistical analyses included one-way ANOVA and Kruskal-Wallis tests, supplemented by post-hoc Tukey HSD tests. Results: A PMMA-HA scaffold with a 20:80 ratio demonstrated the highest antibacterial activity against S. aureus, A. actinomycetemcomitans, P. gingivalis, and F. nucleatum. This was followed by the 30:70 and 40:60 ratios in terms of antibacterial activity. Statistical significance was achieved with p < 0.05 in comparison to controls. However, none of the PMMA-HA ratios showed antifungal activity against C. albicans. Conclusion: PMMA-HA scaffolds have significant activity against bacteria, but not against fungi.

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.

Gingival Mesenchymal Stem Cells Metabolite Decreasing TRAP, NFATc1, and Sclerostin Expression in LPS-Associated Inflammatory Osteolysis In Vivo.

OBJECTIVE: Bone is a dynamic tissue that undergoes remodeling. During bone remodeling, there are transcription factors such as nuclear factor-activated T cells-1 (NFATc1), sclerostin, and tartrate-resistant acid phosphatase (TRAP) that are released for bone resorption. Metabolite from gingival mesenchymal stem cells (GMSCs) has the ability to activate proliferation, migration, immunomodulation, and tissue regeneration of bone cells and tissues. Furthermore, the aim of this study is to investigate the metabolite of GMSCs' effect on expression of NFATc1, TRAP, and sclerostin in calvaria bone resorption of Wistar rats. MATERIALS AND METHODS: Twenty male healthy Wistar rats (Rattus norvegicus), 1 to 2 months old, 250 to 300 g body were divided into four groups, namely group 1 (G1): 100 µg phosphate-buffered saline day 1 to 7; group 2 (G2): 100 µg lipopolysaccharide (LPS) day 1 to 7; group 3 (G3): 100 µg LPS + 100 µg GMSCs metabolite day 1 to 7; and group 4 (G4): 100 µg GMSCs metabolite day 1 to 7. Escherichia coli LPS was used to induce inflammatory osteolysis on the calvaria with subcutaneous injection. GMSCs metabolite was collected after passage 4 to 5, then injected subcutaneously on the calvaria. All samples were sacrificed on the day 8 through cervical dislocation. The expression of TRAP, NFATc1, and sclerostin of osteoclast in the calvaria was observed with 1,000× magnification. STATISTICAL ANALYSIS: One-way analysis of variance and Tukey honest significant different were conducted to analyze differences between groups (p < 0.05). RESULTS: The administration of GMSCs metabolite can significantly decrease TRAP, NFATc1, and sclerostin expression (p < 0.05) in LPS-associated inflammatory osteolysis calvaria in Wistar rats (R. norvegicus). There were significantly different TRAP, NFATc1, and sclerostin expressions between groups (p < 0.05). CONCLUSION: GMSCs metabolite decrease TRAP, NFATc1, and sclerostin expression in LPS-associated osteolysis calvaria in Wistar rats (R. norvegicus) as documented immunohistochemically.

Multidisciplinary Case Management in Mesiodens Impacted Cases with Calcifying Odontogenic Cyst.

Calcifying odontogenic cyst (COC) is a cyst originating from odontogenic epithelium and has a characteristic ghost cell appearance. Clinically, COC is characterized by asymptomatic swelling, leading to lingual expansion, tooth migration, apical resorption, and perforation of cortical bone. COC is most often asymptomatic and is often only discovered during a routine radiological examination. This case report describes the clinical interventions to manage a patient with mesiodens impacted with COC. The procedure is aimed at evaluating the multidisciplinary management of COC at Universitas Airlangga Hospital, Surabaya. Multidisciplinary care has an important role in treating COC cases comprehensively.

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 Identification of Marker Genes for Predicting the Osteogenic Differentiation Potential of Mesenchymal Stromal Cells.

Mesenchymal stromal cells (MSCs) have the potential to differentiate into a variety of mature cell types and are a promising source of regenerative medicine. The success of regenerative medicine using MSCs strongly depends on their differentiation potential. In this study, we sought to identify marker genes for predicting the osteogenic differentiation potential by comparing ilium MSC and fibroblast samples. We measured the mRNA levels of 95 candidate genes in nine ilium MSC and four fibroblast samples before osteogenic induction, and compared them with alkaline phosphatase (ALP) activity as a marker of osteogenic differentiation after induction. We identified 17 genes whose mRNA expression levels positively correlated with ALP activity. The chondrogenic and adipogenic differentiation potentials of jaw MSCs are much lower than those of ilium MSCs, although the osteogenic differentiation potential of jaw MSCs is comparable with that of ilium MSCs. To select markers suitable for predicting the osteogenic differentiation potential, we compared the mRNA levels of the 17 genes in ilium MSCs with those in jaw MSCs. The levels of 7 out of the 17 genes were not substantially different between the jaw and ilium MSCs, while the remaining 10 genes were expressed at significantly lower levels in jaw MSCs than in ilium MSCs. The mRNA levels of the seven similarly expressed genes were also compared with those in fibroblasts, which have little or no osteogenic differentiation potential. Among the seven genes, the mRNA levels of IGF1 and SRGN in all MSCs examined were higher than those in any of the fibroblasts. These results suggest that measuring the mRNA levels of IGF1 and SRGN before osteogenic induction will provide useful information for selecting competent MSCs for regenerative medicine, although the effectiveness of the markers is needed to be confirmed using a large number of MSCs, which have various levels of osteogenic differentiation potential.

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.

Genetic Markers Can Predict Chondrogenic Differentiation Potential in Bone Marrow-Derived Mesenchymal Stromal Cells.

The precise predictions of the differentiation direction and potential of mesenchymal stromal cells (MSCs) are an important key to the success of regenerative medicine. The expression levels of fate-determining genes may provide tools for predicting differentiation potential. The expression levels of 95 candidate marker genes and glycosaminoglycan (GAG) contents after chondrogenic induction in 10 undifferentiated ilium and 5 jaw MSC cultures were determined, and their correlations were analyzed. The expression levels of eight genes before the induction of chondrogenic MSC differentiation were significantly correlated with the GAG levels after induction. Based on correlation patterns, the eight genes were classified into two groups: group 1 genes (AURKB, E2F1, CDKN2D, LIF, and ACLY), related to cell cycle regulation, and group 2 genes (CD74, EFEMP1, and TGM2), involved in chondrogenesis. The expression levels of the group 2 genes were significantly correlated with the ages of the cell donors. The expression levels of CDKN2D, CD74, and TGM2 were >10-fold higher in highly potent MSCs (ilium MSCs) than in MSCs with limited potential (jaw MSCs). Three-dimensional (3D) scatter plot analyses of the expression levels of these genes showed reduced variability between donors and confirmed predictive potential. These data suggest that group 2 genes are involved in age-dependent decreases in the chondrogenic differentiation potential of MSCs, and combined 3D analyses of the expression profiles of three genes, including two group 2 genes, were predictive of MSC differentiation potential.

Characteristic expression of MSX1, MSX2, TBX2 and ENTPD1 in dental pulp cells.

Dental pulp cells (DPCs) are a promising source of transplantable cells in regenerative medicine. However, DPCs have not been fully characterized at the molecular level. The aim of the present study was to distinguish DPCs from various source-derived mesenchymal stem cells (MSCs), fibroblasts (FBs) and other cells by the expression of several DPC-characteristic genes. DPCs were isolated from human pulp tissues by the explant method or the enzyme digestion method, and maintained with media containing 10% serum or 7.5% platelet-rich plasma. RNA was isolated from the cells and from dental pulp tissue specimens. The mRNA levels were determined by DNA microarray and quantitative polymerase chain reaction analyses. The msh homeobox 1, msh homeobox 2, T-box 2 and ectonucleoside triphosphate diphosphohydrolase 1 mRNA levels in DPCs were higher than that of the levels identified in the following cell types: MSCs derived from bone marrow, synovium and adipose tissue; and in cells such as FBs, osteoblasts, adipocytes and chondrocytes. The enhanced expression in DPCs was consistently observed irrespective of donor age, tooth type and culture medium. In addition, these genes were expressed at high levels in dental pulp tissue in vivo. In conclusion, this gene set may be useful in the identification and characterization of DPCs in basic studies and pulp cell-based regeneration therapy.

Age-dependent decrease in the chondrogenic potential of human bone marrow mesenchymal stromal cells expanded with fibroblast growth factor-2.

BACKGROUND AIMS: Human bone marrow mesenchymal stromal cells are useful in regenerative medicine for various diseases, but it remains unclear whether the aging of donors alters the multipotency of these cells. In this study, we examined age-related changes in the chondrogenic, osteogenic and adipogenic potential of mesenchymal stromal cells from 17 donors (25-81 years old), including patients with or without systemic vascular diseases. METHODS: All stem cell lines were expanded with fibroblast growth factor-2 and then exposed to differentiation induction media. The chondrogenic potential was determined from the glycosaminoglycan content and the SOX9, collagen type 2 alpha 1 (COL2A1) and aggrecan (AGG) messenger RNA levels. The osteogenic potential was determined by monitoring the alkaline phosphatase activity and calcium content, and the adipogenic potential was determined from the glycerol-3-phosphate dehydrogenase activity and oil red O staining. RESULTS: Systemic vascular diseases, including arteriosclerosis obliterans and Buerger disease, did not significantly affect the trilineage differentiation potential of the cells. Under these conditions, all chondrocyte markers examined, including the SOX9 messenger RNA level, showed age-related decline, whereas none of the osteoblast or adipocyte markers showed age-dependent changes. CONCLUSIONS: The aging of donors from young adult to elderly selectively decreased the chondrogenic potential of mesenchymal stromal cells. This information will be useful in stromal cell-based therapy for cartilage-related diseases.