Adaptation of Bio-Layer Interferometry for Quantitative Assessment of the Vascular Endothelial Growth Factor Content in Cell-Conditioned Culture Medium

Thixotropic clays have favorable properties for tissue regeneration. Hypoxia mimetic agents showed promising results in pre-clinical models for hard and soft tissue regeneration. It is unclear if clays can be used as carrier for hypoxia mimetic agent in a periodontal regenerative setting. Here, we tested the response of human fibroblasts of the periodontal soft tissue to synthetic clay hydrogels and assessed hypoxia mimetic agent release. Cells were cultured on synthetic clay hydrogels (5.00%–0.15%). We assessed viability and differentiation capacity with resazurin-based toxicity assays, MTT staining, Live-Dead staining, and alkaline phosphatase staining. To reveal the response of fibroblasts to hypoxia mimetic agent-loaded clay hydrogels, cells were exposed to clay supplemented with dimethyloxalylglycine, deferoxamine, l-mimosine, and CoCl2. Supernatants from hypoxia mimetic agent-loaded clay hydrogels were harvested and replaced with medium at hour 1, 3, 6, 24, 48, and 72. To reveal the hypoxia mimetic capacity of supernatants, vascular endothelial growth factor production in the fibroblasts was assessed in the culture medium. Our data show that clay did not induce relevant toxic effects in the fibroblasts which remained capable to differentiate into alkaline phosphatase-positive cells at the relevant concentrations. Fibroblasts cultured on clay hydrogel loaded with dimethyloxalylglycine, deferoxamine, l-mimosine, and CoCl2 remained vital, however, no significant increase in vascular endothelial growth factor levels was found in the culture medium. Only dimethyloxalylglycine-loaded clay supernatants taken in the first hours stimulated vascular endothelial growth factor production in fibroblasts. In conclusion no pronounced toxic effects of synthetic clay were observed. Supplementation with dimethyloxalylglycine leads to hypoxia mimetic activity. This pilot study provides first insights into the impact of synthetic clay on periodontal tissue.

Download Full-text

204 INADEQUACY OF VASCULAR ENDOTHELIAL GROWTH FACTOR IN CULTURE MEDIUM REDUCES THE VIABILITY OF CUMULUS CELLS AND PREVENTS IN VITRO MATURATION OF PORCINE OOCYTES

Reproduction Fertility and Development ◽

10.1071/rdv28n2ab204 ◽

2016 ◽

Vol 28 (2) ◽

pp. 233

Author(s):

T. T. M. Bui ◽

P. P. Ferré ◽

M. T. Tran ◽

T. Wakai ◽

H. Funahashi

Keyword(s):

Vascular Endothelial Growth Factor ◽

Growth Factor ◽

In Vitro Maturation ◽

Culture Medium ◽

Adenosine Monophosphate ◽

Cumulus Cells ◽

Vegf Receptor ◽

Vascular Endothelial ◽

Endothelial Growth Factor

Recently, vascular endothelial growth factor (VEGF) has been regarded as an important factor associated with not only follicle development but also meiotic competence of oocytes. However, the mechanism of how VEGF works is poorly understood. In this study, we investigated in vitro maturation (IVM) of oocytes from different sizes in the absence or presence of a VEGF receptor inhibitor, Axitinib. Cumulus-oocyte complexes (COC) were obtained from small follicles (SF; l < 3 mm in diameter) and medium follicles (MF; 3–6 mm in diameter). Each group of 30–40 COC with at least 3 layers of clear and compact cumulus cells (CC) was cultured in 500 μL of modified porcine oocyte medium (POM-β-mercaptoethanol) supplemented with 10 IU mL–1 eCG, 10 IU mL–1 hCG and 1mM dibutyryl-cyclic-adenosine monophosphate (dbc-AMP) for the first 20 h and then without those supplements for another 24 h at 39°C, 5% CO2 in air. During the first 20 h of IVM, culture medium was also supplemented with or without 1.25 nM Axitinib. At 20 h and 44 h after the start of IVM, the oocytes were denuded and stained with 4′6-diamidino-2-phenylindole (DAPI) to observe the nuclear stages. At 20 h after the start of IVM, some COC were also stained with PI and SYBR Green I to evaluate the ratio of live/dead cumulus cells. Statistical analyses of data from 5 replications were analysed by ANOVA and Tukey’s multiple comparison test. As compared with controls at 20 h after the start of IVM, the number of dead cumulus cells increased significantly in the groups treated with Axitinib, regardless of COC derived from MF and SF (16.8 v. 43.1% in MF and 25.3 v. 57.7% in SF, P < 0.01, respectively). At that time, a majority of oocytes from MF and SF remained at the germinal vesicle (GV) stage in controls (89.8 and 84.6%, respectively), but the percentage significantly reduced in the presence of Axitinib (57.9 and 48.9% of oocytes from MF and SF, respectively) and proceeded around the metaphase-I stage (37.5 and 44.8% of oocytes from MF and SF, respectively). At 44 h after the start of IVM, lower maturation rates were observed in oocytes treated with Axitinib than controls (35.0 v. 81.2% in MF; 20.1 v. 49.0% in SF; P < 0.01). In conclusions, VEGF plays an important role in maitaining the viability of cumulus cells. The presence of VEGFR inhibitor caused the oocytes to develop uncontrollably, even in the presence of dbc-AMP. Moreover, the deficiency of VEGF prevented oocytes fully competent to resume meiosis and arrest to metaphase II.

Download Full-text