Growth factors and their receptors derived from human amniotic cells in vitro

In vitro studies have shown that amnion-produced growth factors participated in angiogenesis, re-epithelialization, and immunomodulation. The aim of our study was to investigate the growth factors and receptors produced by human amnion tissue and amniotic cells. Human amnions (hAM) were isolated, and amnion circles were dissected for in vitro analysis. Some amnion fragments were digested by the use of different methods to obtain two cell fractions, which were analysed for mesenchymal and epithelial cell markers. Amniotic circles and human amniotic cell fractions were cultured in a protein-free medium. Proteins secreted into the culture medium were analysed with a human growth factor antibody array. Conditioned culture media were added to human umbilical vein epithelial cells (HUVECs) to test for stimulation of migration (scratch test) and proliferation (Ki67 expression). Fraction 1 cells expressed both cytokeratin and mesenchymal cell markers which indicated that it was composed of a mixture of human amnion epithelial cells (hAECs) and mesenchymal stromal cells (hAMSCs). Fraction 2 cells mainly expressed cytokeratin and, therefore, were designed as hAECs. Secretion of proteins by the cultured cells increased with time. The hAM cultures secreted EGF-R, IGF, and IGFBP-2,-3 and -6; Cell Fraction 1 secreted NT-4, whereas Cell Fraction 2 secreted G-CSF, M-CSF, and PDGF. Conditioned media of hAM cultures stimulated HUVECs migration. We have showed for the first time that human amnions and amniotic cells secreted IGFBP-6, MCSF-R, PDGF-AB, FGF-6, IGFBP-4, NT-4, and VEGF-R3. We found that Cell Fraction 1, Cell Fraction 2, and the whole amnion secreted different proteins, possibly due to different proportions of amnion-derived cells and different cell-cell interactions. The hAM cell factors remained functional in vitro and induced intensified migration of HUVECs. The growth factors and receptors found in amnion or amniotic cell media might be used for regenerative medicine

The Effect of High and Variable Glucose on the Viability of Endothelial Cells Co-Cultured with Smooth Muscle Cells

Diabetes mellitus causes endothelial dysfunction. The aim of this study was to investigate the effect of normal (5 mmol/L), high (20 mmol/L), and fluctuating (5 and 20 mmol/L changed every day) glucose concentration in the culture medium on the viability of human umbilical vein endothelial cells (HUVECs) co-cultured with human umbilical artery smooth muscle cells (HUASMCs). The cultures were conducted on semi-permeable flat polysulfone (PSU) fibronectin-coated membranes immobilized in self-made inserts. The insert contained either HUVECs on a single membrane or HUASMCs and HUVECs on two membranes close to each other. Cultures were conducted for 7 or 14 days. Apoptosis, mitochondrial potential, and the production of reactive oxygen species and lactate by HUVECs were investigated. The results indicate that fluctuations in glucose concentration have a stronger negative effect on HUVECs viability than constant high glucose concentration. High and fluctuating glucose concentrations slow down cell proliferation compared to the culture carried out in the medium with normal glucose concentration. In conclusion, HUASMCs affect the viability of HUVECs when both types of cells are co-cultured in medium with normal or variable glucose concentration

Wound Area Measurement with Digital Planimetry: Improved Accuracy and Precision with Calibration Based on 2 Rulers

<div><p>Introduction</p><p>In the treatment of chronic wounds the wound surface area change over time is useful parameter in assessment of the applied therapy plan. The more precise the method of wound area measurement the earlier may be identified and changed inappropriate treatment plan. Digital planimetry may be used in wound area measurement and therapy assessment when it is properly used, but the common problem is the camera lens orientation during the taking of a picture. The camera lens axis should be perpendicular to the wound plane, and if it is not, the measured area differ from the true area.</p><p>Results</p><p>Current study shows that the use of 2 rulers placed in parallel below and above the wound for the calibration increases on average 3.8 times the precision of area measurement in comparison to the measurement with one ruler used for calibration. The proposed procedure of calibration increases also 4 times accuracy of area measurement. It was also showed that wound area range and camera type do not influence the precision of area measurement with digital planimetry based on two ruler calibration, however the measurements based on smartphone camera were significantly less accurate than these based on D-SLR or compact cameras. Area measurement on flat surface was more precise with the digital planimetry with 2 rulers than performed with the Visitrak device, the Silhouette Mobile device or the AreaMe software-based method.</p><p>Conclusion</p><p>The calibration in digital planimetry with using 2 rulers remarkably increases precision and accuracy of measurement and therefore should be recommended instead of calibration based on single ruler.</p></div

Box plot of relative errors of area measurement expressed in percentage for the digital planimetry based on one ruler or two ruler calibration techniques.

<p>Box plot of relative errors of area measurement expressed in percentage for the digital planimetry based on one ruler or two ruler calibration techniques.</p

Box plots of relative differences between the measured area and reference area expressed in percentage for the Visitrak device, the Silhouette Mobile device, the AreaMe software and for the digital planimetry methods based on two ruler calibration in 4 ranges of wound area: (A) very small (< 1 cm²), (B) small (1–2 cm²), (C) medium (2–8 cm²), and (D) large (> 8 cm²).

<p>Box plots of relative differences between the measured area and reference area expressed in percentage for the Visitrak device, the Silhouette Mobile device, the AreaMe software and for the digital planimetry methods based on two ruler calibration in 4 ranges of wound area: (A) very small (< 1 cm²), (B) small (1–2 cm²), (C) medium (2–8 cm²), and (D) large (> 8 cm²).</p

Effect of hAM CCM on HUVECs migration assayed by scratch test.

<p>There are results of 160 measurements, 8 independent assays with 10 measurements for test and control each. Median values and (P25, P75) are shown (n = 8, p < 0.05). Detailed description of the assay is in Material and methods.</p

Growth factors in hAM CCM.

<p>Forty-one growth factors were quantitated by antibody array and the obtained values were combined into following growth factor families: EGF family (EGF-2, HB-EGF, EGF-R); FGF family (bFGF, FGF-4, FGF-6, FGF-7); Hematopoietic factors HF (MCSF, MCSF-R, SCF, SCF-R); IGF family (IGF-1, IGF-2, IGF-1SR); IGFBP family (IGFBP-1, IGFBP-2, IGFBP-3, IGFBP-4, IGFBP-6); Neurotrophic factors NF (bNGF, GDNF, NT-3, NT-4); PDGF family (PDGF-AA, PDGF-AB, PDGF-BB, PDGF-Ra, PDGF-Rb); TGF family (TGF-α, TGF-β, TGF-β2, TGF-β3); Vasculogenic factors VF (PLGF, VEGF, VEGF-R3, VEGF-D, VEGF-R2); some growth factors are presented separately: AR; G-CSF; GM-CSF; HGF. Each growth factor fluorescence value (FV) was measured and calculated as described in Materials and methods. (n = 4) (p < 0.05).</p

Effect of hAM CCM on chemotaxy indeks of BM MNCs.

<p>The chemotaxy index (CI) after 2.5 h at 37^°C incubation time was calculated by dividing the number of cells in lower chamber by the number of cells added to the upper chamber counted at the start of the test. Median values and interquartile range (P25, P75) are shown (n = 12, p < 0.05).</p

Effect of hAM CCM on HUVECs expression of cell adhesion molecule-1 (CD31) marker.

<p>Significantly lower expression is observed in presence of hAM CCM. Median values and interquartile ranges (P25, P75) are shown (n = 3, p < 0.05).</p