Cílem tohoto projektu jsou in vitro a in vivo hodnocení nově připravených a charakterizovaných nanokompozitních nosičů se strukturními a mechanickými vlastnostmi optimálními pro osazování mesenchymálními kmenovými buňkami (MSC). Jejich navrhované složení bude kombinovat výhody biodegradabilních alifatických polyesterů ve formě nanovláken, přírodního kolagenu jako matrice, a přírodního kalcium fosfátu ve formě nanočástic, to vše doplněno hyaluronanem sodným. Proces přípravy bude optimalizován s cílem dosažení homogenně porézní mikrostruktury se vzájemně propojenými póry s nano/mikro strukturovaným povrchem, s vhodnými mechanickými vlastnostmi a řízenou dobou biodegradace a to vše s ohledem na získání optimálního systému pro osazování MSC. Výsledky získané pomocí testů in vitro budou využity v testech in vivo na modelu miniaturních prasat.; Newly prepared and characterized nanocomposite scaffolds with suitable structural and mechanical properties for colonization with mesenchymal stem cells promoting the regeneration of defective bone tissue with the required rate of safe biodegradation will be evaluated on the basis of in vitro and in vivo tests. Their proposed composition will combine the advantages of biodegradable polylactide electrospun nanofibers, natural collagen matrix supplemented with sodium hyaluronate and natural calcium phosphate nanoparticles. The preparation process will be optimized, aiming at an interconnected and homogeneously porous material with a nano/microstructured surface, outstanding mechanical properties and a controlled rate of biodegradation capable of withstanding dynamic culture conditions and encouraging homogeneous mesenchymal stem cell (MSC) colonization. This will be tested in vitro, and the acquired knowledge will be transferred to in vivo experiments with miniature pigs.

• MeSH
• fosforečnany vápenaté terapeutické užití   MeSH
• kolagen terapeutické užití   MeSH
• mezenchymální kmenové buňky MeSH
• nanokompozity MeSH
• polyestery terapeutické užití   MeSH
• regenerace kostí MeSH
• testování materiálů MeSH
• vstřebatelné implantáty MeSH

• Konspekt
• Ortopedie. Chirurgie. Oftalmologie
• NLK Obory
• ortopedie
• technika lékařská, zdravotnický materiál a protetika
• NLK Publikační typ
• závěrečné zprávy o řešení grantu AZV MZ ČR

BACKGROUND: Although the highest expression of mutant huntingtin (mtHtt) was observed in the brain, its negative effects were also apparent in other tissues. Specifically, mtHtt impairs metabolic homeostasis and causes transcriptional dysregulation in adipose tissue. Adipogenic differentiation can be induced by the activation of two transcription factors: CCAAT/enhancer-binding protein alpha (CEBPα) and peroxisome proliferator-activated receptor gamma (PPARγ). These same transcription factors were found to be compromised in some tissues of Huntington's disease (HD) mouse models and in lymphocytes of HD patients. OBJECTIVE: This study investigated the adipogenic potential of mesenchymal stem cells (MSCs) derived from transgenic Huntington's disease (TgHD) minipigs expressing human mtHtt (1-548aa) containing 124 glutamines. Two differentiation conditions were used, employing PPARγ agonist rosiglitazone or indomethacin. METHODS: Bone marrow MSCs were isolated from TgHD and WT minipig siblings and compared by their cluster of differentiation using flow cytometry. Their adipogenic potential in vitro was analyzed using quantitative immunofluorescence and western blot analysis of transcription factors and adipogenic markers. RESULTS: Flow cytometry analysis did not reveal any significant difference between WT and TgHD MSCs. Nevertheless, following differentiation into adipocytes, the expression of CEBPα nuclear, PPARγ and adipogenic marker FABP4/AP2 were significantly lower in TgHD cells compared to WT cells. In addition, we proved both rosiglitazone and indomethacin to be efficient for adipogenic differentiation of porcine MSCs, with rosiglitazone showing a better adipogenic profile. CONCLUSIONS: We demonstrated a negative influence of mtHtt on adipogenic differentiation of porcine MSCs in vitro associated with compromised expression of adipogenic transcription factors.

• MeSH
• adipogeneze * MeSH
• buňky kostní dřeně cytologie   MeSH
• geneticky modifikovaná zvířata genetika   MeSH
• Huntingtonova nemoc genetika   patologie   MeSH
• kultivované buňky MeSH
• lidé MeSH
• mezenchymální kmenové buňky cytologie   MeSH
• prasata MeSH
• transkripční faktory genetika   MeSH
• tukové buňky cytologie   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Currently, titanium endosseous implants represent the standard in dental implantology. To improve their mechanical properties, biocompatibility and osseointegration, the concept of bulk nanostructured titanium with variously textured surface is being developed. Different animal species, mostly pigs, minipigs and rabbits, are used for the osseointegration assessment. The selection of implantation site in the target bone is also important, because various types of bones have different ratio between the compact and the trabecular bone tissue. After finishing the in vivo experiment, the implant position and orientation is verified by X-ray imaging. The part of the bone with the implant is surgically removed, cut into tissue blocks, and histologically processed using technique of undemineralized ground sections. This includes embedding the samples into resin followed by sawing, grinding, and polishing of transparent histological sections. A subsequent staining provides information on the formation and mineralization of the bone matrix in close proximity of the implant. Osseointegration is evaluated using a bone-implant contact (BIC) expressed as the ratio of the implant length in contact with bone tissue and total length of the implant. Additional information about the newly formed bone in regions surrounding the implant may be also provided by a double tetracycline test. According to our own experiments, this methodology is simple to perform, highly reproducible, and gives consistent results.

• MeSH
• lidé MeSH
• nanostruktury MeSH
• titan MeSH
• zubní implantáty * MeSH
• zubní materiály MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• práce podpořená grantem MeSH

Quantification of the structure and composition of biomaterials using micro-CT requires image segmentation due to the low contrast and overlapping radioopacity of biological materials. The amount of bias introduced by segmentation procedures is generally unknown. We aim to develop software that generates three-dimensional models of fibrous and porous structures with known volumes, surfaces, lengths, and object counts in fibrous materials and to provide a software tool that calibrates quantitative micro-CT assessments. Virtual image stacks were generated using the newly developed software TeIGen, enabling the simulation of micro-CT scans of unconnected tubes, connected tubes, and porosities. A realistic noise generator was incorporated. Forty image stacks were evaluated using micro-CT, and the error between the true known and estimated data was quantified. Starting with geometric primitives, the error of the numerical estimation of surfaces and volumes was eliminated, thereby enabling the quantification of volumes and surfaces of colliding objects. Analysis of the sensitivity of the thresholding upon parameters of generated testing image sets revealed the effects of decreasing resolution and increasing noise on the accuracy of the micro-CT quantification. The size of the error increased with decreasing resolution when the voxel size exceeded 1/10 of the typical object size, which simulated the effect of the smallest details that could still be reliably quantified. Open-source software for calibrating quantitative micro-CT assessments by producing and saving virtually generated image data sets with known morphometric data was made freely available to researchers involved in morphometry of three-dimensional fibrillar and porous structures in micro-CT scans.

• MeSH
• biokompatibilní materiály analýza   MeSH
• povrchové vlastnosti MeSH
• rentgenová mikrotomografie metody   MeSH
• software MeSH
• testování materiálů * metody   MeSH
• zobrazování trojrozměrné MeSH
• Publikační typ
• práce podpořená grantem MeSH

The study involved the electrospinning of the copolymer poly(L-lactide-co-ε-caprolactone) (PLCL) into tubular grafts. The subsequent material characterization, including micro-computed tomography analysis, revealed a level of porosity of around 70%, with pore sizes of 9.34 ± 0.19 μm and fiber diameters of 5.58 ± 0.10 μm. Unlike fibrous polycaprolactone, the electrospun PLCL copolymer promoted fibroblast and endothelial cell adhesion and proliferation in vitro. Moreover, the regeneration of the vessel wall was detected following implantation and, after six months, the endothelialization of the lumen and the infiltration of arranged smooth muscle cells producing collagen was observed. However, the degradation rate was found to be accelerated in the rabbit animal model. The study was conducted under conditions that reflected the clinical requirements-the prostheses were sutured in the end-to-side fashion and the long-term end point of prosthesis healing was assessed. The regeneration of the vessel wall in terms of endothelialization, smooth cell infiltration and the presence of collagen fibers was observed after six months in vivo. A part of the grafts failed due to the rapid degradation rate of the PLCL copolymer.

• MeSH
• aorta patologie   MeSH
• arteriae carotides patologie   MeSH
• buněčná adheze MeSH
• buňky 3T3 MeSH
• cévní protézy * MeSH
• endoteliální buňky pupečníkové žíly (lidské) MeSH
• endoteliální buňky MeSH
• fibroblasty cytologie   MeSH
• kolagen metabolismus   MeSH
• králíci MeSH
• krysa rodu rattus MeSH
• lidé MeSH
• myocyty hladké svaloviny cytologie   MeSH
• myši MeSH
• polyestery chemie   MeSH
• polymery chemie   MeSH
• poréznost MeSH
• prasata MeSH
• psi MeSH
• regenerace MeSH
• rentgenová mikrotomografie MeSH
• tkáňové inženýrství metody   MeSH
• tkáňové podpůrné struktury MeSH
• transplantace cév * MeSH
• zobrazování trojrozměrné MeSH
• zvířata MeSH
• Check Tag
• králíci MeSH
• krysa rodu rattus MeSH
• lidé MeSH
• myši MeSH
• psi MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

BACKGROUND: Collagen-based scaffolds provide a promising option for the treatment of bone defects. One of the key parameters of such scaffolds consists of porosity, including pore size. However, to date, no agreement has been found with respect to the methodology for pore size evaluation. Since the determination of the exact pore size value is not possible, the comparison of the various methods applied is complicated. Hence, this study focuses on the comparison of two widely-used methods for the characterization of porosity-scanning electron microscopy (SEM) and micro-computed tomography (micro-CT). METHODS: 7 types of collagen-based composite scaffold models were prepared by means of lyophilization and collagen cross-linking. Micro-CT analysis was performed in 3D and in 2D (pore size parameters were: major diameter, mean thickness, biggest inner circle diameter and area-equivalent circle diameter). Afterwards, pore sizes were analyzed in the same specimens by an image analysis of SEM microphotographs. The results were statistically evaluated. The comparison of the various approaches to the evaluation of pore size was based on coefficients of variance and the semi-quantitative assessment of selected qualities (e.g. the potential for direct 3D analysis, whole specimen analysis, non-destructivity). RESULTS: The pore size values differed significantly with respect to the parameters applied. Median values of pore size values were ranging from 20 to 490 µm. The SEM values were approximately 3 times higher than micro-CT 3D values for each specimen. The Mean thickness was the most advantageous micro-CT 2D approach. Coefficient of variance revealed no differences among pore size parameters (except major diameter). The semi-quantitative comparison approach presented pore size parameters in descending order with regard to the advantages thereof as follows: (1) micro-CT 3D, (2) mean thickness and SEM, (3) biggest inner circle diameter, major diameter and area equivalent circle diameter. CONCLUSION: The results indicated that micro-CT 3D evaluation provides the most beneficial overall approach. Micro-CT 2D analysis (mean thickness) is advantageous in terms of its time efficacy. SEM is still considered as gold standard for its widespread use and high resolution. However, exact comparison of pore size analysis in scaffold materials remains a challenge.

• MeSH
• kolagen chemie   MeSH
• poréznost MeSH
• rentgenová mikrotomografie MeSH
• tkáňové inženýrství * MeSH
• tkáňové podpůrné struktury * MeSH
• Publikační typ
• časopisecké články MeSH

The aim of this study was to compare data on the volume fraction of bone and the thickness of the cortical compact bone acquired during microcomputed tomography (micro-CT) analysis with data acquired from identical samples using stereological analysis of either decalcified paraffin sections or ground sections. Additionally, we aimed to compare adjacent tissue samples taken from the major trochanter of the porcine femur to map the basic biological variability of trabecular bone. Fifteen pairs of adjacent tissue blocks were removed from the major trochanter of the proximal epiphyses of porcine femurs (female pigs aged 24-39 months, weight=59.16±8.15kg). In each sample, the volume of the cortical compact bone, the volume of the trabecular bone, and the thickness of the cortical compact bone was assessed using micro-CT. Afterwards, half of the samples were decalcified and processed using paraffin histological sections. Another half was processed into ground sections. The volume and thickness of bone was assessed in histological sections using stereological techniques. There were no significant differences in the bone volumes and thicknesses measured by micro-CT and the corresponding values quantified in decalcified sections. Similarly, there were no differences between the results from micro-CT and the analysis of the corresponding ground sections. Histomorphometric studies based on relatively low numbers of undecalcified ground sections or demineralized paraffin sections of bone yield data on bone volume and the thickness of cortical compact bone that is comparable with three-dimensional micro-CT examination. The pilot data on the variability of cortical compact bone and trabecular bone volumes in the porcine major trochanter provided in this study aim for planning experiments in the field of bone healing and implantology.

• MeSH
• anatomie průřezová MeSH
• dekalcifikace (technika) MeSH
• femur anatomie a histologie   diagnostické zobrazování   MeSH
• fixace tkání metody   MeSH
• kosti a kostní tkáň anatomie a histologie   diagnostické zobrazování   MeSH
• prasata MeSH
• rentgenová mikrotomografie MeSH
• Sus scrofa MeSH
• zalévání tkání do parafínu metody   MeSH
• zvířata MeSH
• Check Tag
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH

Collagen composite scaffolds have been used for a number of studies in tissue engineering. The hydration of such highly porous and hydrophilic structures may influence mechanical behaviour and porosity due to swelling. The differences in physical properties following hydration would represent a significant limiting factor for the seeding, growth and differentiation of cells in vitro and the overall applicability of such hydrophilic materials in vivo. Scaffolds based on collagen matrix, poly(DL-lactide) nanofibers, calcium phosphate particles and sodium hyaluronate with 8 different material compositions were characterised in the dry and hydrated states using X-ray microcomputed tomography, compression tests, hydraulic permeability measurement, degradation tests and infrared spectrometry. Hydration, simulating the conditions of cell seeding and cultivation up to 48 h and 576 h, was found to exert a minor effect on the morphological parameters and permeability. Conversely, hydration had a major statistically significant effect on the mechanical behaviour of all the tested scaffolds. The elastic modulus and compressive strength of all the scaffolds decreased by ~95%. The quantitative results provided confirm the importance of analysing scaffolds in the hydrated rather than the dry state since the former more precisely simulates the real environment for which such materials are designed.

• MeSH
• biokompatibilní materiály chemie   MeSH
• fosforečnany vápenaté chemie   MeSH
• kolagen chemie   MeSH
• kyselina hyaluronová chemie   MeSH
• mechanické jevy MeSH
• modul pružnosti MeSH
• pevnost v tlaku MeSH
• polyestery chemie   MeSH
• poréznost MeSH
• rentgenová mikrotomografie MeSH
• testování materiálů MeSH
• tkáňové inženýrství metody   MeSH
• tkáňové podpůrné struktury chemie   MeSH
• voda chemie   MeSH
• vysoušení * MeSH
• Publikační typ
• časopisecké články MeSH

With the development of a wide range of new biomaterials for the sensing of different cell behaviour, it is important to consider whether the cells tested in vitro are in direct contact with the material or whether cell-biomaterial contact is mediated by an interfacial layer of proteins originating from the culture medium or from the cells themselves. Thus, this study describes the differences between the cell adhesion mediated by proteins originating from foetal bovine serum and without the presence of such proteins 2 h following cell seeding exemplarily with different cell types (an osteoblastic cell line, primary fibroblasts, and mesenchymal stem cells). Three of the examined cell types were found to react differently to differing conditions in terms of cell shape, area, and number. Nevertheless, the expression and localization of the various proteins involved in cell adhesion and signalling (CD44, vinculin, talin, actin, focal adhesion kinase, Rho-GTPases and extracellular signal-regulated kinases 1 and 2) were, in general, similar with respect to all the cell types tested, albeit varying according to the presence or absence of serum. Moreover, no classical focal adhesions were formed during cell adhesion without serum proteins, while different signalling pathways were involved in this process. The study systematically describes and discusses the cell adhesion of three different human cell types to a well-known substrate without the presence of external proteins and it is hoped that this knowledge will be subsequently applied in biomaterial applications in which the presence of external proteins is undesirable (e.g. for biosensing purposes).

• MeSH
• buněčná adheze účinky léků   MeSH
• fibroblasty cytologie   účinky léků   MeSH
• krevní proteiny chemie   farmakologie   MeSH
• kultivované buňky MeSH
• lidé MeSH
• mezenchymální kmenové buňky cytologie   účinky léků   MeSH
• osteoblasty cytologie   účinky léků   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH

Nanocomposite scaffolds which aimed to imitate a bone extracellular matrix were prepared for bone surgery applications. The scaffolds consisted of polylactide electrospun nano/sub-micron fibres, a natural collagen matrix supplemented with sodium hyaluronate and natural calcium phosphate nano-particles (bioapatite). The mechanical properties of the scaffolds were improved by means of three different cross-linking agents: N-(3-dimethylamino propyl)-N'-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide in an ethanol solution (EDC/NHS/EtOH), EDC/NHS in a phosphate buffer saline solution (EDC/NHS/PBS) and genipin. The effect of the various cross-linking conditions on the pore size, structure and mechanical properties of the scaffolds were subsequently studied. In addition, the mass loss, the swelling ratio and the pH of the scaffolds were determined following their immersion in a cell culture medium. Furthermore, the metabolic activity of human mesenchymal stem cells (hMSCs) cultivated in scaffold infusions for 2 and 7 days was assessed. Finally, studies were conducted of cell adhesion, proliferation and penetration into the scaffolds. With regard to the structural stability of the tested scaffolds, it was determined that EDC/NHS/PBS and genipin formed the most effectively cross-linked materials. Moreover, it was discovered that the genipin cross-linked scaffold also provided the best conditions for hMSC cultivation. In addition, the infusions from all the scaffolds were found to be non-cytotoxic. Thus, the genipin and EDC/NHS/PBS cross-linked scaffolds can be considered to be promising biomaterials for further in vivo testing and bone surgery applications.

• MeSH
• analýza selhání vybavení MeSH
• biokompatibilní materiály chemická syntéza   MeSH
• buněčná adheze fyziologie   MeSH
• design vybavení MeSH
• kolagen chemie   MeSH
• kostní matrix chemie   MeSH
• kostní náhrady chemická syntéza   MeSH
• lidé MeSH
• mezenchymální kmenové buňky cytologie   fyziologie   MeSH
• nanokompozity chemie   ultrastruktura   MeSH
• proliferace buněk fyziologie   MeSH
• reagencia zkříženě vázaná chemie   MeSH
• testování materiálů MeSH
• tkáňové podpůrné struktury * MeSH
• transplantace mezenchymálních kmenových buněk přístrojové vybavení   metody   MeSH
• velikost částic MeSH
• viabilita buněk fyziologie   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• hodnotící studie MeSH
• práce podpořená grantem MeSH
• srovnávací studie MeSH