Passage Number of 4T1 Cells Influences the Development of Tumour and the Progression of Metastasis in 4T1 Orthotopic Mice.

Background: The study was aimed to elucidate the influence of passage number of 4T1 cells for the development of the ideal tumour model. Methods: A total of 24 female BALB/c mice was divided equally into three groups: i) control (phosphate buffered saline [PBS] only); ii) group A (subjected to 4T1 cells of passage number 9) and iii) group B (subjected to 4T1 cells of passage number 10). The injections were introduced at the 3rd mammary pad of the mice. The net volume of the tumours was examined. Histopathological analysis was conducted to compare the extent of metastasis in the different groups of mice. Results: Group B had a higher net volume of 4T1 tumour as compared to group A (P = 0.042). The coefficient of variation in the net volume of 4T1 tumour for group A was higher (135.3%) as compared to group B (40.79%). Group A only exhibited metastasis on the lungs, liver and spleen whereas group B showed metastasis to the heart, spleen, lungs and liver. Conclusion: The use of 4T1 cells from passage number 10 is more ideal for the development of 4T1 tumour.

Analysis of serum and gene expression profile of cytokines (IL-6, TNF-α and TGF-ß1) in chronic hepatitis C virus infection.

Background: Chronic hepatitis C virus (HCV) infection is one of the major causes of liver cirrhosis and liver carcinoma. Studies have indicated that an imbalance of cytokine activities could contribute to the pathogenesis of chronic HCV infection. This study aimed to investigate serum levels and gene expression of cytokines (IL-6, TNF-α and TGF-ß1) in chronic HCV infection among Malay male subjects. Methods: Thirty-nine subjects were enrolled from various health clinics in Kelantan, Malaysia, and divided into two groups: patients with chronic HCV infection (HP) and healthy control (HS). The serum cytokines IL-6, TNF-a-were measured using Luminex assay, and serum TGF-ß1 was measured by ELISA. The mRNA gene expression for IL-6, TNF-α and TGF-ß1 was measured by real-time reverse transcriptase polymerase chain reaction (RT-PCR). Results: There were statistically significant differences in the mean serum levels of IL-6, and TGF-ß1 in HP compared to HS group (p = 0.0180 and p = 0.0005, respectively). There was no significant difference in the mean serum level of TNF-α in HP compared to HS group. The gene expression for the studied cytokines showed no significant differences in HP compared to HS group. Conclusion: Serum IL-6 was significantly associated with chronic HCV infection.

The influence of macrophage polarisation status and IL-6 in breast carcinoma invasion / La influencia del estado de polarización de los macrófagos y la IL-6 en la invasión del carcinoma de mama

Introduction: Breast cancer metastasis accounts for the majority of deaths from breast cancer. The knowledge on IL-6 affecting cancer cell metatastatic behaviour need to be studied.Objectives: This study aim to examine the association of macrophage polarisation status and IL-6 with clinicopathological criteria and lymphovascular invasion (LVI) of breast carcinoma.Material & Method: 81 cases of FFPE breast carcinoma samples were stained with IL6, CD80 (M1 macrophage), CD204 and CD163 (M2 macrophage) and CD68 (pan-macrophage marker). The macrophages count were evaluated based on 3 hotspots of positively stained cells. IL-6 scoring was done using the H-score method.Result: Significant association was observed between CD68 marker with blood vessel invasion (p-value = 0.014), lymphatic vessel invasion (p-value = 0.005), and metastasis (p-value =0.028). CD68 was also significantly associated with CD204 (p = 0.027). CD80 biomarker also showing significant association with patient tumour grade (p-value = 0.054), ER (0.028) and PR (0.010) in patient clinical data and CD204 is significantly associated with ER (0.053) and PR (0.054) patient clinical data. Meanhile, there is no significant association of IL-6 with the patient clinical data.Conclusion: There is no significant association of IL-6 with the patient clinicopathological data obtained in this study while CD68 showed significant correlation with M2 macrophage biomarker and LVI indicating the influence of M1 and M2 macrophage in breast cancer metastatic pathway through blood and lymphatic vessel invasion. (AU)

Introducción: La metástasis del cáncer de mama representa la mayoría de las muertes por cáncer de mama. Es necesario estudiar el conocimiento sobre la IL-6 que afecta el comportamiento metatastásico de las células cancerosas.Objetivos: Este estudio tiene como objetivo examinar la asociación del estado de polarización de los macrófagos y la IL-6 con los criterios clínico-patológicos y la invasión linfovascular (LVI) del carcinoma de mama.Material y método: 81 casos de muestras de carcinoma de mama FFPE se tiñeron con IL6, CD80 (macrófago M1), CD204 y CD163 (macrófago M2) y CD68 (marcador pan-macrófago). El recuento de macrófagos se evaluó en base a 3 hotspots de células teñidas positivamente. La puntuación de IL-6 se realizó mediante el método de puntuación H.Resultado: Se observó una asociación significativa entre el marcador CD68 con invasión de vasos sanguíneos (valour de p = 0,014), invasión de vasos linfáticos (valour de p = 0,005) y metástasis (valour de p = 0,028). CD68 también se asoció significativamente con CD204 (p = 0.027). El biomarcador CD80 también muestra una asociación significativa con el grado del tumour del paciente (valour p = 0,054), ER (0,028) y PR (0,010) en los datos clínicos del paciente y el CD204 se asocia significativamente con los datos clínicos del paciente ER (0,053) y PR (0,054). Mientras tanto, no existe una asociación significativa de IL-6 con los datos clínicos del paciente.Conclusión: No existe una asociación significativa de IL-6 con los datos clínico-patológicos del paciente obtenidos en este estudio, mientras que CD68 mostró una correlación significativa con el biomarcador de macrófagos M2 y LVI que indica la influencia de los macrófagos M1 y M2 en la vía metastásica del cáncer de mama a través de la invasión de vasos sanguíneos y linfáticos. (AU)

Extracellular Vesicles from Mesenchymal Stem Cells as Potential Treatments for Osteoarthritis.

Osteoarthritis (OA) is a chronic degenerative disorder of the joint and its prevalence and severity is increasing owing to ageing of the population. Osteoarthritis is characterized by the degradation of articular cartilage and remodeling of the underlying bone. There is little understanding of the cellular and molecular processes involved in pathophysiology of OA. Currently the treatment for OA is limited to painkillers and anti-inflammatory drugs, which only treat the symptoms. Some patients may also undergo surgical procedures to replace the damaged joints. Extracellular vesicles (EV) play an important role in intercellular communications and their concentration is elevated in the joints of OA patients, although their mechanism is unclear. Extracellular vesicles are naturally released by cells and they carry their origin cell information to be delivered to target cells. On the other hand, mesenchymal stem cells (MSCs) are highly proliferative and have a great potential in cartilage regeneration. In this review, we provide an overview of the current OA treatments and their limitations. We also discuss the role of EV in OA pathophysiology. Finally, we highlight the therapeutic potential of MSC-derived EV in OA and their challenges.

Optimization of scanning electron microscope technique for amniotic membrane investigation: A preliminary study.

OBJECTIVE: The objective of this study was to evaluate and compare the two scanning electron microscope (SEM) preparation protocols and determine the better SEM preparation technique to study stem cells on human amniotic membrane (hAM) scaffold. MATERIALS AND METHODS: Formaldehyde-based protocol and glutaraldehyde-based protocol were compared to evaluate the quality of SEM images for stem cells cultured on hAM scaffold. RESULTS: The results suggested that formaldehyde-based protocol is better than glutaraldehyde-based protocol in terms of showing clearer topography of the membrane as well as the boarders of the cells. To provide intact surface of the SEM sample and avoid possible ruptures of the hAM or the thin cell layer, it is recommended to perform the dehydration step using graded alcohol concentrations of 20%, 30%, 40%, 50%, 60%, 70%, 80%, and 90%, one time for each and twice in 100% for 10 min each. Gold sputter-coating step is not recommended as it does not improve the image quality. CONCLUSIONS: To obtain clear SEM images, it is recommended to run a preliminary study to determine the better chemicals and conditions of sample preparation even when following preexisting protocols.

Gypsum-based biomaterials: Evaluation of physical and mechanical properties, cellular effects and its potential as a pulp liner.

This in vitro study aimed to evaluate setting time and compressive strength of gypsum-based chitosan biomaterials and its effect on proliferation of stem cells from human exfoliated deciduous teeth (SHED) and alkaline phosphatase (ALP) activity. Pure-GYP was mixed with water (2.5 g: 1.9 mL); Gyp-CHT was prepared with gypsum, chitosan, and water (2.5 g: 0.285 g: 1.9 mL). Cell viability and ALP activity were assessed at different periods. Data were analyzed using SPSS (p<0.05). The setting times were 2.7 min and 2.8 min for pure-GYP and Gyp-CHT, respectively. Significantly higher compressive strength was observed with Gyp-CHT. SHED treatments with both materials were not cytotoxic. ALP was consistently higher in the treated groups compared with the control. Cellular attachments were evident with SEM. Excellent cellular viability with pure-GYP and Gyp-CHT, as well as increased ALP activities, suggested the possibility of tertiary dentin formation. Further studies are necessary to evaluate the biomaterials for its pulp protective potentialities.

Synergistic effects of chitosan scaffold and TGFß1 on the proliferation and osteogenic differentiation of dental pulp stem cells derived from human exfoliated deciduous teeth.

OBJECTIVE: Multipotent stem cells derived from human exfoliated deciduous teeth (SHED) represent a promising cell source for tissue regeneration. In the present study we decided to test the inductive effect of chitosan and transforming growth factor-ß1 (TGFß1) as a scaffold/factor combination on SHED proliferation and osteogenic differentiation. DESIGN: Cell proliferation was quantitatively assessed by PrestoBlue, live/dead assay was performed and cell attachment to chitosan scaffold was examined by scanning electron microscopy (SEM). For osteogenic differentiation analysis, alkaline phosphatase activity was quantified, cells were stained with Alizarin Red, and the lineage specific genes/proteins ALP, COL I, BSP, and OCN were analysed by real-time PCR and Western blot. RESULTS: SHED remained viable and attached well to the chitosan structure. Moreover, TGFß1 significantly enhanced the proliferative activity of SHED on the chitosan scaffold. Our data further revealed that chitosan and TGFß1 enhanced the osteogenic differentiation of SHED, as evidenced by high ALP activity, strong mineral deposition, and the up-regulation of ALP, COL I, BSP, and OCN gene/protein expression. CONCLUSION: Together, data from our study indicate that the combination of chitosan scaffolds and TGFß1 enhanced proliferation and osteogenic differentiation of SHED. These findings suggest that the combined application of chitosan scaffold and TGFß1 in conjunction with SHED might be beneficial for in vivo bone regeneration.

Isolation and enhancement of a homogenous in vitro human Hertwig's epithelial root sheath cell population.

Hertwig's epithelial root sheath (HERS) cells play a pivotal role during root formation of the tooth and are able to form cementum-like tissue. The aim of the present study was to establish a HERS cell line for molecular and biochemical studies using a selective digestion method. Selective digestion was performed by the application of trypsin-EDTA for 2 min, which led to the detachment of fibroblast-like-cells, with the rounded cells attached to the culture plate. The HERS cells displayed a typical cuboidal/squamous-shaped appearance. Characterization of the HERS cells using immunofluorescence staining and flow cytometry analysis showed that these cells expressed pan-cytokeratin, E-cadherin, and p63 as epithelial markers. Moreover, RT-PCR confirmed that these cells expressed epithelial-related genes, such as cytokeratin 14, E-cadherin, and ΔNp63. Additionally, HERS cells showed low expression of CD44 and CD105 with absence of CD34 and amelogenin expressions. In conclusion, HERS cells have been successfully isolated using a selective digestion method, thus enabling future studies on the roles of these cells in the formation of cementum-like tissue in vitro.