The effect of preconditioning hypoxia in schwann-like-cells-derived adipose mesenchymal stem cells and rat sciatic nerve-derived stem cells: experimental research.

The preconditioning hypoxia for stem cells is a strategy to achieve effective conditions for cell therapy, indicate increased expression of regenerative genes in stem cell therapy, and enhance the secretion of bioactive factors and therapeutic potential of their cultured secretome. Objectives: This study aims to explore the response of Schwann-like cells derived from adipose-derived mesenchymal stem cells (SLCs) and Schwann cells rat sciatic nerve-derived stem cells (SCs) with their secretomes under normoxic and hypoxic conditions in vitro. Material and methods: SLCs and SCs were isolated from the adipose tissue and the sciatic nerve of the adult white male rat strain Wistar. Cells were incubated in 21% O2 (normoxic group) and 1%, 3%, and 5% O2 (hypoxic group) conditions. Concentration values of transforming growth factor-ß (TGF-ß), basic Fibroblast Growth factor (bFGF), brain-derived neurotrophic factor, glial-derived neurotrophic factor, vascular endothelial growth factor, and nerve growth factor were detected and calculated utilizing an enzyme-linked immunosorbent assay, and the growth curve was described. Results: SLCs and SCs indicated positive expression for mesenchymal markers and negative expression for hematopoietic markers. Normoxic conditions SLCs and SCs showed elongated and flattened morphology. Under hypoxic conditions, SLCs and SCs showed a classic fibroblast-like morphology. Hypoxia 1% gave the highest concentration in TGF-ß and bFGF from the SLCs group and TGF-ß, bFGF, brain-derived neurotrophic factor, and vascular endothelial growth factor from the SCs group. No significant differences in concentration of growth factors between the SLCs group compared to SCs group in all oxygen groups. Conclusions: Preconditioning hypoxia has an effect on the composing of SLCs, SCs, and their secretomes in vitro; no significant differences in concentration of growth factors between the SLCs group compared with the SCs group in all oxygen groups.

Computational approach in searching for dual action multitarget inhibitors for osteosarcoma.

Osteosarcoma is a common primary malignant bone tumor that typically manifests in the second decade of life. This study aimed to identify osteogenic compounds that potentially serve as multitarget inhibitors for osteosarcoma. The study was a molecular docking study of nine Food and Drug Administration-approved compounds with osteogenic properties to the key membrane proteins of osteosarcoma. The ligands used were raloxifene, simvastatin, dexamethasone, risedronate, ibandronate, zoledronic acid, ascorbic acid, alendronate, and ß-glycerophosphate, whereas the target proteins used were RET, fibroblast growth factor receptor 1, KIT, PDGFRA, VEGFR1, and VEGFR2. Chem3D version 15.0.0.106 was used for ligand preparation, and AutoDockTools version 1.5.6 was used for protein preparation, whereas molecular docking was conducted using AutoDock Vina. Raloxifene, simvastatin, and dexamethasone had the lowest binding activity to the target proteins. The binding affinity of raloxifene was from -8.4 to -10.0 kcal mol-1, that of simvastatin was -8.3 to -9.2 kcal mol-1, whereas dexamethasone ranged from -6.9 to -9.1 kcal mol-1. Most types of interactions were hydrophobically followed by hydrogen bonding. The current study suggests that raloxifene, simvastatin, and dexamethasone have the potential to act as multitarget inhibitors for osteosarcoma with the ability to induce bone remodeling.

The efficacy of topical adipose mesenchymal stem cell-conditioned medium versus framycetin gauze dressing in chronic plantar ulcer of leprosy: A randomized controlled trial.

Background Wound healing shows a unique interaction of several cells, growth factors and cytokines. The healing of chronic plantar ulcer of leprosy is influenced by various factors, one of which is the concentration of growth factors and cytokines related to the pathogenesis of impaired wound healing. Growth factors and cytokines can be found in the secretome of adipose mesenchymal stem cells. Aim To compare the effectiveness of topical adipose mesenchymal stem cell-conditioned medium and framycetin gauze dressing only on the healing of chronic plantar ulcer of leprosy. Methods In this randomised controlled trial, 32 patients with chronic plantar ulcer of leprosy were recruited. After detailed clinical and initial debridement, patients were randomised to two groups to receive either topical adipose mesenchymal stem cell-conditioned medium (n = 16) or framycetin gauze dressing only (n = 16) applied every three days for up to eight weeks, following which the ulcer size, adverse reactions and complications if any were monitored weekly. Results Healing percentage increased each week in all groups. Statistical differences between groups (P < 0.05) were observed from week 2 onwards for ulcer mean size reduction and from week 3 onwards for ulcer mean depth reduction. There were no adverse reactions or complications. Limitations Off-loading on subjects were not performed. Conclusion Adipose mesenchymal stem cell-conditioned medium is a potential therapeutic agent in the management of chronic plantar ulcer of leprosy.

Alveolar Repair Using Cancellous Bone and Beta Tricalcium Phosphate Seeded With Adipose-Derived Stem Cell.

INTRODUCTION: Adipose-derived stem cells (ADSCs) have been subject of several studies due to their abundance, ease of preparation, and application in bone regeneration. We aim to compare effectiveness of alveolar reconstruction utilizing human cancellous freeze-dried graft (HCG) and beta tricalcium phosphate (BTP), both seeded with human ADSC (hADSC) and autologous bone graft (ABG). MATERIAL AND METHODS: A 5 × 5 mm alveolar defect in 36 male Wistar rats were treated using: ABG (C), HCG-hADSC (H1), and BTP-hADSC (H2). At 1 and 8 weeks after surgery, runt-related transcription factor 2 (RUNX2), alkaline phosphatase (ALP), osterix (OSX), and bone morphogenetic protein 2 (BMP2; g/mL) were quantified using immunohistochemistry, while bone tissue volume (BV, mm3), bone tissue volume fraction (BF, percentage), and trabecular thickness of bone (TT, mm) were assessed using micro-computed tomography (CT). RESULTS: One week after surgery, H2 was higher in RUNX2, OSX, ALP, and BMP2 than C (P < .05). Only RUNX2 and OSX were found to be higher in H1 than C, while ALP and BMP2 were higher in H2 than H1. Micro-CT revealed that H2 had a higher TT than C and C had a higher TT than H1 (P < .05). Eight weeks after surgery, both H2 and H1 was higher in RUNX2, OSX, ALP, and BMP2 than C (P < .05). RUNX2 and BMP2 were found to be higher in H1 than H2. Micro-CT revealed that H2 had higher BV and TT than C and H1 (P < .05). CONCLUSIONS: Exogenous hADSC strengthened the effectiveness of HCG and BTP to accelerate osteogenesis, osteoconduction, and osteoinduction. The latter was the most successful in bone formation, followed by HCG and ABG.

Prospect of Stem Cells as Promising Therapy for Brachial Plexus Injury: A Systematic Review.

Background: Brachial plexus injury is an advanced and devastating neurological injury, for which both nerve surgery and tendon transfers sometimes remain insufficient in restoring normal movement. Stem cell therapy may be applicable to rescue the injured motor neurons from degeneration which potentially improves muscle strength. Study Design: Systematic Review; Level of evidence V. Data Sources: A systematic literature search was conducted on PubMed (MEDLINE), EMBASE, the Cochrane Library, and Scopus using the terms ("stem cell") AND ("brachial plexus") as search keywords. Methods: The process of study selection was summarized by PRISMA flow diagram. The study included in vivo and in vitro studies with English language, humans or animals with some brachial plexus injuries, interventions, some applications of stem cells to the groups of study, with functional, biomechanical, or safety outcomes. Results: In total, there were 199 studies identified from the literature sources where 75 articles were qualified for forward evaluation following selecting the titles and abstracts. Ten studies were finally included in this systematic review after full-text assessment. Stem cells can produce neurotrophic factors in vitro and in vivo in rats, and their level was increased after injury. Electrophysiological measurement showed that the intervention group had distinctly higher CMAP amplitude and evidently shorter CMAP latency than the model group. Application of bone marrow stem cells (BMSCs) showed an elevation in the numbers of axons and density of myelinated fibers, the density of nerve fibers, the diameter of regenerating axons, and a decrease in axonal degeneration. A study in humans indicated an improvement of the movements in a patient with traumatic total BPI after injection of Ad-MSC. It is associated with increased muscle mass and sensory recovery and also suggested that mononuclear cell injection enhances muscle regeneration and reinnervation in the partly denervated muscle of brachial plexus injury. Various muscle groups had obtained strength together with restoration, the muscle strength attained after the previous transplantation were preserved. The results of this review support stem cell treatment in brachial plexus injury. Conclusion: This review provides evidence of the positive effects of stem cell treatment in brachial plexus injury.

An update of current therapeutic approach for Intervertebral Disc Degeneration: A review article.

Intervertebral disc degeneration is a natural process of aging. It can cause physical, psychological, and socioeconomic impact due to the decreasing function of the spine and pain manifestation. Conservative and surgical treatment to correct symptoms and structural anomalies does not fully recover the degenerated disc. Several therapeutic approaches have been developed to improve the clinical result and patient's quality of life. This paper aims to review previous studies that discussed potential novel approach in order to make effective degenerated disc restoration. We tried to briefly describe IVD, IDD, also review several promising current therapeutic approaches for degenerated disc treatment, including its relevance to the degeneration process and limitation to be applied in a clinical setting. There are generally four current therapeutic approaches that we reviewed; growth factors, small molecules, gene therapy, and stem cells. These new approaches aim to not only correct the symptoms but also restore and delay the degeneration process.

Does freeze dried amnion useful as scaffold for mesenchymal stem cell for repair of vesicovaginal fistula? An overview of proliferative and remodeling phase in the wound healing process.

BACKGROUND: Surgery is still the main solution for vesicovaginal fistula, but postoperative wound healing represents a challenge, and the recurrence rate remains high. There is a need to develop therapeutic methods to increase the success of therapy and women's quality of life. OBJECTIVE: To explore whether human freeze-dried amnion is useful as a mesenchymal stem cell scaffold for repair of vesicovaginal fistula through assessment of the proliferative and remodelling phases. METHODS: This experiment was undertaken using a New Zealand rabbit model. The research was divided into two stages: (1) an experiment to create a model of a vesicovaginal fistula; and (2) a laboratory experiment to close a vesicovaginal fistula as a result of the first stage. The second stage used a post-test-only control group design. The wound-healing process was assessed based on the expression of platelet-derived growth factor (PDGF), vascular endothelial growth factor (VEGF), fibroblast growth factor (FGF), occludin and claudin-4. Data were analysed descriptively and statistically. RESULTS: Expression of PDGF, VEGF, FGF, occludin and claudin-4 in vesicovaginal fistula models sutured with human freeze-dried amnion was higher compared with models without human freeze-dried amnion. Significant differences were found in average expression of PDGF, VEGF, FGF, occludin and claudin-4. CONCLUSION: Human freeze-dried amnion plays a role in the wound-healing process in vesicovaginal fistula repair models. It is hoped that this research will improve urogynaecological services.

Effects of bone marrow mesenchymal stem cell transplantation on tumor necrosis factor-alpha receptor 1 expression, granulosa cell apoptosis, and folliculogenesis repair in endometriosis mouse models.

BACKGROUND AND AIM: Endometriosis affects the ovaries and causes a decrease in the oocyte quality during endometrial receptivity. During the development of ovarian follicles, paracrine communication occurs between granulosa cells and oocytes. This study was conducted to determine the effects of bone marrow mesenchymal stem cell transplantation on tumor necrosis factor-alpha (TNF-α) receptor 1 (TNFR1) expression, granulosa cell apoptosis, and folliculogenesis in endometriosis mouse models. MATERIALS AND METHODS: This study involved 42 female mice, which were divided into three groups: Healthy mice (T0), endometriosis mice without transplantation (T1), and endometriosis mice with bone marrow mesenchymal stem cell transplantation (T2). The mice were injected intraperitoneally with endometrial fragments (200 µL) to become endometriosis models. On day 15, the endometriosis models received mesenchymal stem cells. Sample collection was performed on day 29. Granulosa cell apoptosis and TNFR1 expression were examined using immunohistochemical staining, and folliculogenesis was assessed using hematoxylin and eosin staining of ovary samples. The data obtained from both examinations were statistically analyzed using Statistical Package for the Social Sciences. RESULTS: The results showed that TNFR1 expression is significantly decreased in T2 (p<0.004). The apoptosis of granulosa cells was lower in T2 (p<0.000). The primary, secondary, and graafian follicle counts in T2 were significantly increased. CONCLUSION: Bone marrow mesenchymal stem cell transplantation in endometriosis mouse models can reduce TNFR1 expression and granulosa cell apoptosis and improve folliculogenesis.

The Escalation of Osteosarcoma Stem Cells Apoptosis After the Co-Cultivation of Peripheral Blood Mononuclear Cells Sensitized with Mesenchymal Stem Cells Secretome and Colony Stimulating Factor-2 in vitro.

INTRODUCTION: Peripheral blood mononuclear cells (PBMCs) sensitized with mesenchymal stem cells (MSCs) secretome and/or colony stimulating factor-2 (CSF-2) as an immunotherapy candidate may escalate osteosarcoma stem cells (OS-SCs) apoptosis. This study aimed to investigate the escalation of osteosarcoma stem cells' apoptosis after the co-cultivation with PBMCs sensitized by MSCs secretome with/or CSF-2 and it was completed by analyzing the level of serum tumor necrosis factor-related apoptosis-inducing ligand (sTRAIL) and tumor necrosis factor-α (TNF-α) level, annexin V binding, caspase-3 and caspase-8 expression in vitro. METHODS: OS-SCs were derived from a single human osteosarcoma sample with its high grade and osteoblastic essential clinical characteristics obtained from a biopsy before the chemotherapy treatment. They were then isolated and cultured confirmed by the cluster of differentiation-133 (FITC) by applying immunofluorescence analysis with fluorescein isothiocyanate (FITC) labeled. MSCs secretome was obtained with cells extracted from the bone marrow of a healthy patient. Furthermore, enzyme linked immunosorbent assay (ELISA) was utilized to analyze sTRAIL and TNF-α level in each group. The expression of caspase-3, caspase-8, and annexin V assay in each group was examined by applying the immunofluorescence labeled with FITC. The comparison analysis between treatment groups and the control group was performed by utilizing the analysis of variance (ANOVA) and continued with Tukey Honest Significant Difference (HSD) (p<0.05). RESULTS: There was a significant difference in the upregulation of sTRAIL and TNF-α level indicated by the increased annexin V, caspase-3, and caspase-8 expression binding between groups (p<0.05). CONCLUSION: MSCs Secretome and CSF-2 could significantly increase the activity of PBMCs through the improvement of sTRAIL and TNF-α levels which could lead to the escalation of OS-SCs apoptosis through an enhanced expression of caspase 3, caspase 8 and annexin V binding in vitro.

Role of adipose mesenchymal stem cells and secretome in peripheral nerve regeneration.

The use of stem cells is a breakthrough in medical biotechnology which brings regenerative therapy into a new era. Over the past several decades, stem cells had been widely used as regenerative therapy and Mesenchymal Stem Cells (MSCs) had emerged as a promising therapeutic option. Currently stem cells are effective therapeutic agents againts several diseases due to their tissue protective and repair mechanisms. This therapeutic effect is largely due to the biomolecular properties including secretomes. Injury to peripheral nerves has significant health and economic consequences, and no surgical procedure can completely restore sensory and motor function. Stem cell therapy in peripheral nerve injury is an important future intervention to achieve the best clinical outcome improvement. Adipose mesenchymal stem cells (AdMSCs) are multipotent mesenchymal stem cells which are similar to bone marrow-derived mesenchymal stem cells (BM-MSCs). The following review aims to provide an overview of the use of AdMSCs and their secretomes in regenerating peripheral nerves.

Gingival-Derived Mesenchymal Stem Cell from Rabbit (Oryctolagus cuniculus): Isolation, Culture, and Characterization.

OBJECTIVE: This study aims to confirm whether the GDMSCs isolated from rabbit's (Oryctolagus cuniculus) gingiva are mesenchymal stem cells (MSCs). MATERIALS AND METHODS: This study design was partly quasi-experimental with an observational design. GDMSCs were isolated from the gingiva of healthy male rabbits (O. cuniculus) (n = 2), 6 months old, and 3 to 5 kg of body weight. The specific cell surface markers of MSCs; clusters of differentiation (CD), namely, CD44, CD73, CD90, CD105, and CD200 expressions; and hematopoietic stem cell surface markers CD34 and CD45 were examined using flow cytometry and immunohistochemistry with immunofluorescence. The osteogenic differentiation of isolated GDMSCs was examined using alizarin red staining. RESULTS: GDMSCs in the fourth passage showed a spindle-like formation and fibroblast-like cells that attached to the base of the culture plate. GDMSCs were MSCs that positively expressed CD44, CD73, CD90, CD105, and CD200 but did not express CD34 and CD45 when examined using flow cytometry and immunohistochemical analysis. GDMSCs had osteogenic differentiation confirmed by calcified deposits in vitro with a red-violet and brownish color after alizarin red staining. CONCLUSION: GDMSCs isolated from the rabbits (O. cuniculus) were confirmed as MSCs in vitro documented using immunohistochemistry and flow cytometry. GDMSCs can differentiate into osteogenic lineage in vitro that may be suitable for regenerative dentistry.

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.

The enhancement apoptosis of osteosarcoma mesenchymal stem cells co-cultivation with peripheral blood mononuclear cells sensitized by secretome and granulocyte macrophage colony-stimulating factor.

The advanced, metastasis, and reccurent of osteosarcoma (OS) patients have a poor prognosis postaggresive surgery and chemotherapy. Peripheral blood mononuclear cells (PBMCs) as cell-based immunotherapy may successful in the OS treatment. To investigate the enhancement apoptosis of OS-mesenchymal stem cells (OS-MSCs) co-cultivated with PBMCs sensitized using the secretome and granulocyte macrophage colony-stimulating factor (GMCSF). This true experimental study with posttest only control group design and in vitro study. The sample was cultured OS-MSCs which confirmed by Cluster of Differentiation-133 using immunocytochemistry (ICC) and histopathology analysis. The sample divided into six groups accordingly: OS-MSC, OS-MSC + PMBC, OS-MSC + PMBC + Secretome, OS-MSC + PMBC + GMCSF, OS-MSC + PBMC + Secretome + GMCSF (n = 5/N = 30). The enhancement of OS-MSCs apoptosis was analyzed through Interleukin-2 (IL-2) level through the Enyzme-Linked Immunosorbent Assay examination, expression of Signal Transducers and Activators of Transcription (STAT)-3 and caspase-3 by ICC. One-way analysis of variance test and Tukey Honestly Significant Difference to analyze the difference between the groups (P < 0.05). The highest of IL-2 level was found in the PBMC + Secretome + GMCSF group. The highest expression of caspase-3 was found in OS-MSC + PBMC + Secretome + GMCSF group with significant different between groups (P < 0.05). There was insignificant difference of STAT-3 epxression and IL-2 level between groups (P > 0.05). The co-cultivation of OS-MSCs and PBMSCs activated using secretome and GMCSF has a great ability to enhance OS-MSCs apoptosis.

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 potency of chitosan-based Pinus merkusii bark extract nanoparticles as anti-cancer on HeLa cell lines.

BACKGROUND AND AIM: Cervical cancer accounts for the fourth as a cause of death from cancer in women worldwide, with more than 85% of events and deaths occurring in developing countries. The main problems of chemotherapy are the lack of selectivity and drug resistance. This study aimed to investigate the signal transduction of chitosan-based Pinus merkusii bark extract nanoparticles (Nano-PMBE) as an anticancer on HeLa cell line. MATERIALS AND METHODS: Nano-PMBE was prepared based on the ionic gelation method. Its anticancer activities in HeLa cells were investigated through cytotoxicity test, cell cycle, and apoptosis analysis. The expression of p53 and caspase-9 was also observed. RESULTS: The results showed that Nano-PMBE has a size of 394.3 nm. Meanwhile, the Nano-PMBE was cytotoxic to HeLa cells (IC50 of 384.10 µg/ml), caused G0/G1 phase arrest and cell apoptosis in HeLa cells. Besides, the expression of p53 and caspase-9 has increased. CONCLUSION: The results showed a notable anticancer effect of Nano-PMBE by arresting the cell cycle and inducing apoptosis in HeLa cells, suggesting that it might have therapeutic potential for cervical cancer. Further research is needed to find out more about the anticancer mechanism of Nano-PMBE in HeLa cells to in vivo and clinical studies.

In vitro anticancer activity Annona squamosa extract nanoparticle on WiDr cells.

This study aimed to prepare Annona squamosa leaf extract-loaded chitosan nanoparticles (nano-ASLE) against human colon cancer (WiDr) cells. Nano-ASLE was made with ionic gelation method. Four concentrations of the nano-ASLE (50, 100, 200, and 400 µg/mL) in dimethyl sulfoxide were prepared on WiDr cells to determine the IC50 value using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Then, it was divided into three groups of concentration of IC50, 2IC50, and 4IC50 and continued with analysis of caspase-3 expression and cell cycle arrest. The results of particles size were obtained 535.1 nm and showed potent cytotoxicity with IC50 292.39 µg/mL. The expression of caspase-3 increased significantly and caused cell cycle arrest at the G2/M phase and induced apoptosis on WiDr cells. Further studies are needed to obtain the loading efficiency, release of drug concentration, and in vivo study of nano-ASLE to suppress WiDr cells.

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.

Osteogenic potential of gingival stromal progenitor cells cultured in platelet rich fibrin is predicted by core-binding factor subunit-α1/Sox9 expression ratio ( in vitro).

Background: Alveolar bone defect regeneration has long been problematic in the field of dentistry. Gingival stromal progenitor cells (GSPCs) offer a promising solution for alveolar bone regeneration. In order to optimally differentiate and proliferate progenitor cells, growth factors (GFs) are required. Platelet rich fibrin (PRF) has many GFs and can be easily manufactured. Core-binding factor subunit-α1 (CBF-α1) constitutes a well-known osteogenic differentiation transcription factor in SPCs. Sox9, as a chondrogenic transcription factor, interacts and inhibits CBF-α1, but its precise role in direct in vitro osteogenesis remains unknown. GSPCs cultured in vitro in PRF to optimally stimulate osteogenic differentiation has been largely overlooked. The aim of this study was to analyze GSPCs cultured in PRF osteogenic differentiation predicted by CBF-α1/Sox9. Methods: This study used a true experimental with post-test only control group design and random sampling. GPSCs isolated from the lower gingiva of four healthy, 250-gram, 1-month old, male Wistar rats ( Rattus Novergicus) were cultured for two weeks, passaged every 4-5 days. GSPCs in passage 3-5 were cultured in five M24 plates (N=108; n=6/group) for Day 7, Day 14, and Day 21 in three different mediums (control negative group: αModified Eagle Medium; control positive group: High Glucose-Dulbecco's Modified Eagle Medium (DMEM-HG) + osteogenic medium; Treatment group: DMEM-HG + osteogenic medium + PRF). CBF-α1 and Sox9 were examined with ICC monoclonal antibody. A one-way ANOVA continued with Tukey HSD test (p<0.05) based on Kolmogorov-Smirnov and Levene's tests (p>0.05) was performed. Results: The treatment group showed the highest CBF-α1/Sox9 ratio (16.00±3.000/14.33±2.517) on Day 7, while the lowest CBF-α1/Sox9 ratio (3.33±1.528/3.67±1.155) occurred in the control negative group on Day 21, with significant difference between the groups (p<0.05). Conclusion: GSPCs cultured in PRF had potential osteogenic differentiation ability predicted by the CBF-α1/sox9 ratio.

Hypoxic preconditioning effect on stromal cells derived factor-1 and C-X-C chemokine receptor type 4 expression in Wistar rat's (Rattus norvegicus) bone marrow mesenchymal stem cells (in vitro study).

AIM: To examine the effect of hypoxic preconditions on the ability of bone marrow stem cells culture mediated expression C-X-C chemokine receptor type 4 (CXCR4) and stromal cells derived factor-1 (SDF-1) in vitro. MATERIALS AND METHODS: Bone marrow mesenchymal stem cells (BMSCs) were derived from 12 femurs of 200 g Wistar male rats. The animals were euthanized before BMSCs isolation. BMSCs were divided into two groups, control group: Normoxic condition 21% O2 and treatment group: Hypoxic condition 1% O2. The characterization of BMSCs was analyzed using flow cytometry by cluster differentiation 34 and cluster differentiation 105. The expression of CXCR4 and SDF-1 measured using immunocytochemistry immunofluorescence label after 48-h incubation in a low-tension oxygen chamber with an internal atmosphere consisting of 95% N2, 5% CO2, and 1% O2. All data were subjected to a normality test and then analyzed using t-test statistic (p<0.05). RESULTS: The characterization of bone marrow stem cells showed positive cluster differentiation 34 and cluster differentiation 105. A hypoxic precondition (1% O2) in culture increases CXCR4 (p=0.000) and SDF-1 expression than normoxic conditions (p=0.000) (p<0.05). CONCLUSION: Hypoxic preconditioning with 1% O2 increase CXCR4 and SDF1 expression.