2023-01-29
22:31 |
Mineralised polylactide and polycaprolactone soft foams with hierarchical micro-macro porous structure for tissue engineering
Beran, M. ; Berková, E. ; Musílková, Jana ; Sedlář, Antonín ; Slepička, P. ; Fajstavr, D.
The purpose of the study was development of soft foams from resorbable polymers with unique micro-macro porous interconnected hierarchical structure specially designed as scaffold for engineering of soft tissues. The foams have been prepared by freeze-drying of solutions of polylactide (PLA) and polycaprolactone (PCL) in 1,4-dioxane. The foams prepared by freeze-drying had interconnected porous aerogel characteristics. The hierarchical structure with bimodal micro-macro pore size distribution were obtained after addition of sucrose or NaCl porogens with defined crystal size distributions to the solutions before freeze-drying and leaching the porogen crystals from the freeze-dried foams with demineralised water. Polyethyleneimine was chemically conjugated to the alkali-treated foams followed by conjugation of citric acid using carbodiimide chemistry. Finally, they were mineralised by immersing and incubating in a simulated body fluid with ionic concentration similar to that of human blood plasma, to obtain tissue engineering scaffolds. To verify their biocompatibility, the scaffolds were seeded with adipose-derived stem cells (ASC) and sarcoma osteogenic-2 (SaOs-2) human osteoblast-like cells. Morphology of the cells attached to the scaffolds was evaluated and their viability was verified by a metabolic test. Biocompatibility and usability of the scaffolds was successfully verified by incubation with adipose-derived stem cells and SaOs-2 human osteosarcoma cell line. Mineralised scaffolds are more suitable growth supports for both the cell types than unmineralized collagen scaffolds. The scaffolds have been specially designed for engineering of soft tissues, but they can be used in other categories of tissue engineering, too.
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2023-01-29
22:31 |
The co-culture of keratinocytes and fibroblasts on a multi-layered polyester nanofibrous membrane enriched with platelet lysate
Blanquer, Andreu ; Filová, Elena ; Jenčová, V. ; Brož, Antonín ; Kuželová Košťáková, E. ; Lisnenko, M. ; Procházková, R. ; Bačáková, Lucie
The prevalence of chronic wounds is increasing due to the population ageing and specific illnesses like diabetes mellitus and vascular diseases. Nanofibrous membranes fabricated using synthetic polymers are promising materials to enhance skin wound healing. PCL and PVA membranes are being studied to be used as scaffolds for skin tissue engineering and hydrogels for controlled drug delivery, respectively. The present study considers the development of a multi-layered membrane made of PCL and PVA loaded with platelet lysate (PL). PCL nanofibers allowed cell adhesion and growth, whereas PVA acted as a hydrogel that releases the bioactive compounds of platelet lysate. The cytocompatibility of the membranes containing PL and without it was demonstrated on two cell types involved in wound healing, i.e. keratinocytes and fibroblasts. Both cell types were able to adhere and proliferate on the membranes. In addition, the membrane containing PL enhanced the proliferation of fibroblasts. A co-culture study was also performed by seeding each cell type on one side of the membrane. The cells were co-cultured for 7 days and the results showed that PL increased the proliferation of cells achieving a monolayer of keratinocytes or fibroblasts on each side of the membrane. Thus, the beneficial effect of PCL-PVA+PL membranes on monocultures and co-cultures of skin cells was demonstrated, and these membranes can be considered potential scaffolds for treatment of chronic wounds.
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2023-01-29
22:31 |
Differentiation of mesenchymal stem cells on fibrin assemblies supported by immobilized growth factors FGF2 and VEGF
Musílková, Jana ; Filová, Elena ; Kaplan, Ondřej ; Bačáková, Lucie
Bioartificial heart valves and vascular grafts prepared from decellularized tissues could be recellularized with bone marrow-derived mesenchymal stem cells (MSCs) that are able to differentiate into both smooth muscle cells and endothelial cells. MSCs differentiation is facilitated by sustained release of growth factors. In our study assemblies based on fibrin, fibrin with heparin, fibrin with adsorbed or covalently-immobilized vascular endothelial growth factor A165 (VEGF) or basic fibroblast growth factor (FGF-2) via binding to heparin attached to fibrin have been prepared and were evaluated for their stimulation of MSCs differentiation. We estimated the mRNA expression of endothelial marker CD31 (PECAM1), smooth muscle marker α-actin (ACTA2), osteoblast markers osteocalcin (BGLAP) and alkaline phosphatase (ALP). The gene expression was estimated using RT-PCR on days 1, 7 and 21 after seeding. The cell morphology and viability was evaluated by LIVE/DEAD staining. VEGF, both adsorbed and covalently bound, increased significantly the expression of smooth muscle marker α-actin. The mRNA expression of ACTA2 on day 7 and 21 raised more than 200 times in comparison to control samples (undifferentiated cells before seeding). The ACTA2 gene expression significantly exceeded the expression of all other evaluated genes at all time intervals. Moreover, on day 21, the late smooth muscle marker desmin (DES) was steeply rising in cells cultivated on assemblies containing heparin and covalently bound VEGF. The expression of osteocalcin was minimal. We conclude that fibrin assembly containing covalently bound VEGF is the most convenient for MSCs differentiation towards smooth muscle cells.
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2019-10-19
16:35 |
Adhesion and growth of adipose tissue-derived stem cells on fibrin assemblies with attached growth factors for tissue engineering of heart valves
Filová, Elena ; Trávníčková, Martina ; Knitlová, Jarmila ; Matějka, Roman ; Kučerová, Johanka ; Riedelová, Zuzana ; Brynda, Eduard ; Bačáková, Lucie
Currently used xenogeneic biological heart valve prostheses are decellularized and crosslinked with glutaraldehyde. These grafts usually undergo degeneration and calcification. Pericardium-based heart valve prostheses, re-seeded with autologous cells, i.e. Adipose tissue-derived cells (ASCs) and endothelial cells, could have longer durability and biocompatibility. In order to improve the adhesion of cells and their ingrowth into decellularized pericardium, various fibrin (Fb) layers were developed, i.e. Fb, Fb with covalently bound heparin (H), Fb with either vascular endothelial growth factor (VEGF) or fibroblast growth factor 2 (FGF) in various concentrations (1 ng/ml, 10 ng/ml, 100 ng/ml) or with both VEGF and FGF (100 ng/ml). Growth factors were attached onto Fb via heparin or were adsorbed. ASCs were seeded on theses layers in a DMEM medium supplemented with 2 % of fetal bovine serum, TGFβ1 and BMP-4 (both 2.5 ng/ml), and with ascorbic acid. Cell adhesion and growth/viability was assessed by counted cell number/MTS evaluation. ASCs were stained for differentiation markers of smooth muscle cells, such as alpha-actin, calponin, and myosin heavy chain. On day 7, ASCs on Fb-H-VEGF layers produced both calponin and alpha-actin. An increased FGF concentration caused reduced calponin staining of ASCs. Lack of heparin in fibrin assemblies with growth factors inhibited the production of both alpha-actin and calponin in ASCs. The highest ASCs density/viability was found on Fb-H-VEGF-FGF layer. The proper formulation of fibrin coatings could be favorable for ASCs growth and differentiation and could subsequently support endothelialization of cardiovascular prostheses with endothelial cells.
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2018-04-06
16:29 |
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2017-03-10
16:12 |
Odborná zpráva o postupu prací a dosažených výsledcích za rok 2016 v rámci projektu TA04011345
Hrůzová, D. ; Matějka, R. ; Rosina, J. ; Zárubová, Jana ; Filová, Elena
Zpráva shrnuje postupy realizace projektu TA04011345 Cévní protézy o malém průměru osídlované endotelovými a kmenovými buňkami kostní dřeně v bioreaktoru v rámci etapy 2016. Hlavními body jsou: optimalizace decelularizačního postupu pro získání standardizovaného materiálu pro buněčné osídlení, postup trojrozměrného osídlování cévní protézy - endotelizace, vývoj systému bioreaktoru, mechanické zkoušky, chemické potahování protézy, získání buněk od pacientů a jejich charakterizace.
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2017-03-10
16:12 |
Analysis of high-frequency activity in electroencephalogram of animal epilepsy model
Havel, T. ; Kudláček, J. ; Jiruška, Přemysl ; Ježdík, P. ; Čmejla, R.
High frequency oscillations (HFO) are believed to be a new specific biomarker of epileptogenic tissue. According to the reported findings, we hypothesized that HFOs occur more specific to the epileptogenic tissue. To test this hypothesis, we analyzed intracranial electroencephalograms (iEEG) of three subjects of animal epilepsy model. Overall 180 minutes of iEEG records were processed by the automatic high frequency activity detector and further analyzed. The dominant frequency of each segment was determined and categorized as ripples (80-200 Hz) or fast ripples (200-1000 Hz). Even though overall number of HFO detections predominate in the hemisphere where epileptic focus is located, in comparison of each brain structure separately our hypothesis cannot be fully confirmed.
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2017-03-10
16:12 |
Case study of intracranial EEG records of patients with focal cortical dysplasia type I and II
Balach, J. ; Ježdík, P. ; Čmejla, R. ; Kršek, P. ; Jiruška, Přemysl
In this study we try to find out if it is possible to differentiate type of focal cortical dysplasia by features obtained from intracranial EEG. We compare occurrence and rates of three biomarkers present in epilepsy in patients with focal cortical dysplasia type I and II. Case study is made on long term night records of 6 pediatric patients. Detection of interictal epileptiform discharges and high-frequency oscillations is made by automated algorithms, delta brush are marked visually. Position of lesion and electrodes inside were obtained from MRI. In individual rates were not found difference on significant level. No major significance were found, but as promising seem to be ratio inside to outside rates of high-frequency oscillations and presence of delta brush, which were found only in patients with focal cortical dysplasia type II.
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2017-02-07
15:49 |
Growth of human adipose-derived stem cells on Ti-6Al-4V alloy with various surface modifications
Kročilová, Nikola ; Bačáková, Lucie ; Pařízek, Martin ; Havlíková, Jana ; Motarjemi, H. ; Molitor, M. ; Gabor, R. ; Marvan, J.
Adipose-derived stem cells (ASCs) are of a great importance for bone tissue engineering. We compared ASCs obtained by liposuction under two pressures (-200 mmHg and -700 mmHg). The number and proliferation activity of cells isolated under -700 mmHg (ASC-H) were higher than in cells isolated under -200 mmHg (ASC-L). However, the ASC-L was more active in osteogenic differentiation, as manifested by a higher intensity of fluorescence of alkaline phosphatase and osteocalcin in these cells. The adhesion and growth of ASCs were then studied on Ti-6Al-4V samples either unmodified (K, roughness parameter R-a = 280 nm) or modified by shot blasting and tarnishing A, R-a = 200 nm), vibratory finishing (B, R-a = 100 nm) and vibratory finishing, shot blasting and polishing (C, R-a = 80 nm). All modified samples were more wettable than the K samples. On day 1 after seeding, the size of cell spreading area on some modified samples was lower than on K samples, namely on B samples in ASC-L, on B and C in ASC-H and on A in control MG-63 cells. On day 3, the cell number on some modified samples became higher than on K samples, namely on C samples (ASC-L), on B (ASC-H) and on B and C s (MG-63 cells). On day 7, the numbers of ASC-H and MG-63 cells on all modified samples evened out and became significantly higher than on K samples. On ASC-L samples, the highest cell numbers were obtained on A samples. Thus, all studied modifications of Ti-6Al-4V enhanced growth of ASCs and human osteoblast-like MG-63 cells.
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2017-02-07
15:49 |
Growth and differentiation of human osteoblast-like cells on TI-6AL-4V alloy modified with anodization
Doubková, Martina ; Bačáková, Lucie ; Pařízek, Martin ; Vandrovcová, Marta ; Musílková, Jana ; Lisá, Věra ; Gabor, R. ; Marvan, J.
Titanium and its alloys are widely used as hard tissue implants for their high biocompatibility and suitable physical properties. Along with other surface treatment methods, anodization technique is known to enhance osseointegration. The aim of this study is to evaluate the adhesion, growth and osteogenic differentiation of human osteoblast-like SAOS-2 cells on Ti-6Al-4V samples anodized in electrolytes composed of a stable volume of KOH (336.48 g/l) and variable volumes of liquid glass (124.47 g/l, 84,12 g/l and 37.38 g/l; samples s5, s6 and s7, respectively). Non-anodized Ti-6Al-4V samples, cell culture polystyrene (PS) and microscopic glass coverslips served as control materials. On days 2 and 4 after seeding, the cell number did not differ significantly among the tested samples. However, on day 7, the cell number on s6 samples reached the lowest values, which could be attributed to a non-homogeneous TiO2 film on s6 samples formed during anodization. Nevertheless, the osteogenic differentiation, estimated by the intensity of fluorescence of collagen I in cells grown in a differentiation medium, was the highest on s6 samples. On s5 samples, coated with homogeneous TiO2 films, both cell numbers and intensity of fluorescence of collagen I was relatively high. The bone matrix mineralization, evaluated by Alizarin Red staining, was the highest on s5 samples in standard culture medium, and similar on all tested samples in differentiation medium. Thus, the surface modification of s5 samples could be considered the most suitable for application in bone implants.
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