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

Cell-Free DNA Analysis of Epithelial Growth Factor Receptor Mutations in Lung Adenocarcinoma Patients by Droplet Digital PCR

Cell-free DNA (cfDNA) analysis may provide a non-invasive diagnostic approach for lung adenocarcinoma patients. Recently, droplet digital PCR (ddPCR) has been developed as a highly sensitive detection method for a low mutant allele percentage. The ddPCR detection limit for epithelial growth factor receptor (EGFR) mutations was evaluated using cell lines, NCI-H1975 for EGFR L858R point mutation and PC-9 for EGFR E746-A750del. Subsequently, detection of EGFR mutations by ddPCR was performed in tumor DNA (tDNA) and cfDNA samples of 19 lung adenocarcinoma patients whose tumor biopsies were already evaluated for EGFR mutations by clamp PCR (13 of L858R, 3 of E746-750del, and 3 of EGFR negative). In 12 cases, immunohistochemical analysis was performed to quantify the number of EGFR L858R-positive cells rate. EGFR point mutation or deletion were detected in 16 tumor DNA samples. In the measurable cfDNA samples, the rate of detection by ddPCR in cfDNA was 61.5% (8/13) for L858R and 100% (3/3) for E746-A750del. A relative correlation was found between the allele fraction (AF) of tDNA and the number of EGFR L858R-positive cells rate. No correlation was found between the AF of tDNA and AF of cfDNA. In our study, cfDNA mutation detection was not associated with clinicopathological features, but cases with high AF of cfDNA did have metastatic lesions. Our study shows that ddPCR enables cfDNA analysis for EGFR L858R and E746-A750del, with a high detection rate. Therefore, cfDNA analysis using ddPCR may complement to tumor biopsy and is beneficial for precision medicine in lung adenocarcinoma patients

Differential Expression Levels of Plasma microRNAs in Neuroblastoma Patients Identified by Next-Generation Sequencing

Efforts to identify biomarkers for neuroblastoma (NB) have been ongoing, but no definite biomarker has been identified in peripheral blood. We proposed the use of plasma exosomal miRNAs as biomarkers of unfavorable NB patient outcomes. Exosomal miRNAs isolated from 31 plasma and 37 tissue samples, many from the same NB patients, were sequenced using a next-generation sequencing instrument. We analyzed the correlation between miRNA expression levels in plasma and tissue samples with International Neuroblastoma Risk Group staging system (INRGSS) outcome and MYCN status. We chose differentially expressed miRNAs with similar expression patterns in plasma and tissue samples in each of the three analysis groups and combined those miRNAs to find the optimal combination with the potential to be considered as a biomarker. MicroRNA-92a-3p was found to be significantly upregulated in deceased patients (p = 0.017), miR-375 was upregulated in INRGSS stage M patients (p = 0.002), and plasma miR-92a-3p and miR-99a-5 levels were upregulated in patients with MYCN amplification (p = 0.007 and 0.006). The combination of miR-92a-3p, miR-375, and miR-99a-5p levels was shown to be a statistically significant predictor of NB patient outcomes (AUC = 0.726, p = 0.001, 95% CI = 0.612–0.841, sensitivity = 77%, specificity = 56.7%). Thus, the combination of miR-92a3p, miR-375, and miR-99a-5p may potentially be used as a biomarker for unfavorable NB patient outcomes. However, further validation is required in a larger number of NB patients.This research was partially supported by a Grant-in-Aid for Scientific Research (A) (No.19H0105600) from the Ministry of Education, Culture, Sports, Science and Technology

Potential Marker Genes for Predicting Adipogenic Differentiation of Mesenchymal Stromal Cells

Mesenchymal stromal cells (MSCs) are a promising source for tissue engineering of soft connective tissues. However, the differentiation capacity of MSCs varies among individual cell lines. Here, we show marker genes to predict the adipogenic potential of MSCs. To clarify the correlation between gene expression patterns before adipogenic induction and the differentiation level of MSCs after differentiation, we compared mRNA levels of 95 genes and glycerol-3-phosphate dehydrogenase (GPDH) activities in 15 MSC lines (five jaw and 10 ilium MSCs) from 15 donors. Expression profiles of 22 genes before differentiation significantly correlated with GPDH activities after differentiation. Expression levels of 11 out of the 22 genes in highly potent ilium MSCs were at least three times higher compared with jaw MSCs, which have limited differentiation potential. Furthermore, three-dimensional scatter plot for mRNA expression of ITGA5, CDKN2D, and CD74 could completely distinguish highly potent MSCs from poorly potent MSCs for adipogenesis. The treatment of MSC cultures with the anti-ITGA5 antibody reduced adipogenic differentiation of MSCs. Collectively, these results suggest that the three genes play a role in adipogenesis before induction and can serve as predictors to select potent MSCs for adipogenic differentiation

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

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

Mixed Polymethylmethacrylate and Hydroxyapatite as a Candidate of Synthetic Graft Materials for Cleft Palate

This research aimed to investigate scaffolds of mixed polymethylmethacrylate (PMMA) and hydroxyapatite (HA) that can be considered as a candidate of synthetic graft materials for cleft palate. Polymethylmethacrylate-hydroxyapatite (PMMA-HA) scaffold was done by weighing PMMA granules and HA powder for a 20:80, 30:70 and 40:60 ratio, that mixed and moulded in 5 mm of diameter and 10 mm of height, then freeze dried before being analyzed by X-Ray Diffraction (XRD), Fourier Transform Infra-Red (FTIR), Scanning electron microscope (SEM) and Energy dispersive X-ray (EDX) instruments. XRD analysis in 20:80, 30:70 and 40:60 ratio has a peak that represents HA and an amorphous peak that represents PMMA, with the best ratio is 30:70.FTIR analysis showed the existence of HA functional groups namely phosphate (PO43-), calcium carbonate (CO32-), and hydroxyl (OH-) in all three samples, with 20:80 ratio as the optimal composition. SEM observation showed visible interconnected pores and roughness, also various pore sizes that averaged by 278.71µm; 312.3µm; 332.8µm, and 1.267µm; 1.716µm; 1.951µm, that showed by 50x and 5000x magnification, with 20:80 ratio as the ideal environment. EDX analysis showed Calsium (Ca), Oxygen (O), Phosphate (P), Silicate (Si), and Aluminium (Al) contained in the PMMA-HA sample, with 20:80 ratio as the highest potention to osteoblast regeneration. PMMA-HA scaffold with 20:80 ratio considered as the best ratio to be a candidate of synthetic graft materials for cleft palate

Characterization of human dental pulp cells grown in chemically defined serum‑free medium

Dental pulp cells (DPCs) are promising candidates for use as transplantable cells in regenerative medicine. However, ex vivo expansion of these cells typically requires culture media containing fetal bovine serum, which may cause infection and immunological reaction following transplantation. In addition, the proliferation and differentiation of DPCs markedly depend upon serum batches. Therefore, the present study examined whether DPCs could be expanded under serum-free conditions. DPCs obtained from four donors were identified to proliferate actively in the serum-free medium, STK2, when compared with those cells in control medium (Dulbecco's modified Eagle's medium containing 10% serum). The high proliferative potential with STK2 was maintained through multiple successive culture passages. DNA microarray analyses demonstrated that the gene expression profile of DPCs grown in STK2 was similar to that of cells grown in the control medium; however, a number of genes related to cell proliferation, including placental growth factor and inhibin-βE, were upregulated in the STK2 cultures. Following induction of osteogenesis, DPCs grown in STK2 induced alkaline phosphatase activity and calcification at higher levels compared with the control medium cultures, indicating maintenance of differentiation potential in STK2. This serum-free culture system with DPCs may have applications in further experimental studies and as a clinical strategy in regenerative medicine

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

الملخص: أهداف البحث: الحنك المشقوق هو عيب خلقي شائع يتطلب غالبا ترقيع العظام السنخية للحصول على علاج فعال. كان الهدف من هذه الدراسة هو دراسة الأنشطة المضادة للبكتيريا والفطريات لمختلط بولي ميثيل ميثاكريلات-هيدروكسيباتيت ضد الكائنات الحية الدقيقة في الفم. يمكن أن تفيد النتائج الاستخدام المحتمل لـ بولي ميثيل ميثاكريلات-هيدروكسيباتيت كمواد تطعيم عظمية اصطناعية لعلاج عيوب العظام السنخية في حالات الحنك المشقوق. طريقة البحث: تم تحضير مسحوق هيدروكسيباتيت من مركز السيراميك في إندونيسيا وحبيبات بولي ميثيل ميثاكريلات من مختبرات هاي ميديا بنسبة 20:80 و30:70 و40:60. تم استخدام طريقة الانتشار المضاد للبكتيريا ضد المكورات العنقودية الذهبية، المشعشعة المصاحبة للورم الفطري، وحيدات الخلية البورفيرينية اللثوية و المغزلية الناخرة، في حين تم اختبار طريقة الانتشار المضاد للفطريات ضد المبيضات البيضاء. استخدمت بروتوكولات موحدة للزراعة الميكروبية. تم قياس مناطق التثبيط باستخدام الفرجار الرقمي. وشملت التحليلات الإحصائية اختبارات أنوفا و كروسكال-واليس أحادية الاتجاه، بالإضافة إلى اختبارات ''تركي الفرق الكبير بصراحة'' اللاحقة. النتائج: أظهرت سقالة بولي ميثيل ميثاكريلات-هيدروكسيباتيت بنسبة 20:80 أعلى نشاط مضاد للجراثيم ضد المكورات العنقودية الذهبية، المشعشعة المصاحبة للورم الفطري، وحيدات الخلية البورفيرينية اللثوية و المغزلية الناخرة. وتلاها النسب 30:70 و40:60 من حيث النشاط المضاد للبكتيريا. تم تحقيق أهمية إحصائية بالمقارنة مع الضوابط. ومع ذلك، لم تظهر أي من نسب بولي ميثيل ميثاكريلات-هيدروكسيباتيت نشاطا مضادا للفطريات ضد المبيضات البيضاء. الاستنتاجات: سقالات بولي ميثيل ميثاكريلات-هيدروكسيباتيت لها نشاط مضاد للجراثيم، ولكن ليس لها نشاط مضاد للفطريات. Abstract: 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 Staphylococcus aureus, 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