The development of an ideal vascular prosthesis represents an important challenge in terms of the treatment of cardiovascular diseases with respect to which new materials are being considered that have produced promising results following testing in animal models. This study focuses on nanofibrous polycaprolactone-based grafts assessed by means of histological techniques 10 days and 6 months following suturing as a replacement for the rat aorta. A novel stereological approach for the assessment of cellular distribution within the graft thickness was developed. The cellularization of the thickness of the graft was found to be homogeneous after 10 days and to have changed after 6 months, at which time the majority of cells was discovered in the inner layer where the regeneration of the vessel wall was found to have occurred. Six months following implantation, the endothelialization of the graft lumen was complete, and no vasa vasorum were found to be present. Newly formed tissue resembling native elastic arteries with concentric layers composed of smooth muscle cells, collagen, and elastin was found in the implanted polycaprolactone-based grafts. Moreover, the inner layer of the graft was seen to have developed structural similarities to the regular aortic wall. The grafts appeared to be well tolerated, and no severe adverse reaction was recorded with the exception of one case of cartilaginous metaplasia close to the junctional suture.
- Publikační typ
- časopisecké články MeSH
Úvod: Kardiovaskulární choroby jsou zodpovědné za významnou morbiditu i mortalitu ve společnosti. Užití umělých cévních materiálů je často nezbytnou součástí v rámci chirurgické léčby, ať již je tato radikální nebo paliativní. V současné době dochází k vývoji řady nových biodegradabilních materiálů určených pro tyto účely. Preklinické testování každého nového materiálu je naprosto nezbytné, je prováděno jak in vitro, tak in vivo. Z tohoto důvodu jsou zvířecí experimentální modely nadále nutnou součástí testování před klinickým užitím. Cílem této práce je prezentovat možnosti užití různých zvířecích modelů na poli kardiovaskulární chirurgie a jejich extrapolace do klinické medicíny. Metody: Autoři prezentují jejich obecné zkušenosti s experimentální chirurgií, na jejich podkladě diskutují optimální výběr zvířecího modelu pro testování nových materiálů pro kardiovaskulární chirurgii a stejně tak optimální lokalitu implantace. Výsledky: Jako optimální experimentální zvířecí modely pro testování hemokompatibility a degradability nových materiálů uvádějí autoři modely potkana, králíka a prasete. Intraperitoneální implantace u potkana je snadná a lehce proveditelná procedura, stejně tak jako arteriální bandáž na aortě králíka či prasete. Rovněž karotické tepny jsou dobře využitelné. Bandáž na prasečí pulmonální tepně je již složitější zákrok s četnějšími komplikacemi. Explantované bandážované cévy po předem definované době jsou vhodné pro další mechanické testování ve smyslu biomechanických analýz, např. inflačně-extenzního testu. Závěr: V posledních fázích preklinického testování nových materiálů se nelze nadále obejít bez in-vivo experimentů. Naší snahou je však striktně dodržovat koncept 3R – Replacement, Reduction a Refinement. V tomto smyslu je třeba využít co nejvíce potenciál každého zvířete tak, abychom mohli redukovat počty zvířat.
Introduction: Cardiovascular diseases are responsible for significant morbidity and mortality in the population. Artificial vascular grafts are often essential for surgical procedures in radical or palliative treatment. Many new biodegradable materials are currently under development. Preclinical testing of each new material is imperative, both in vitro and in vivo, and therefore animal experiments are still a necessary part of the testing process before any clinical use. The aim of this paper is to present the options of using various experimental animal models in the field of cardiovascular surgery including their extrapolation to clinical medicine. Methods: The authors present their general experience in the field of experimental surgery. They discuss the selection process of an optimal experimental animal model to test foreign materials for cardiovascular surgery and of an optimal region for implantation. Results: The authors present rat, rabbit and porcine models as optimal experimental animals for material hemocompatibility and degradability testing. Intraperitoneal implantation in the rat is a simple and feasible procedure, as well as aortic banding in the rabbit or pig. The carotid arteries can also be used, as well. Porcine pulmonary artery banding is slightly more difficult with potential complications. The banded vessels, explanted after a defined time period, are suitable for further mechanical testing using biomechanical analyses, for example, the inflation-extension test. Conclusion: An in vivo experiment cannot be avoided in the last phases of preclinical research of new materials. However, we try to strictly observe the 3R concept – Replacement, Reduction and Refinement; in line with this concept, the potential of each animal should be used as much as possible to reduce the number of animals.
BACKGROUND/AIM: Anastomotic leakage is a feared complication in colorectal surgery. Postoperative peritoneal adhesions can also cause life-threatening conditions. Nanofibrous materials showed their pro-healing properties in various studies. The aim of the study was to evaluate the impact of double-layered nanofibrous materials on anastomotic healing and peritoneal adhesions formation. MATERIALS AND METHODS: Two versions of double-layered materials from polycaprolactone and polyvinyl alcohol were applied on defective anastomosis on the small intestine of healthy pigs. The control group remained with uncovered defect. Tissue specimens were subjected to histological analysis and adhesion scoring after 3 weeks of observation. RESULTS: The wound healing was inferior in the experimental groups, however, no anastomotic leakage was observed and the applied material always kept covering the defect. The extent of adhesions was larger in the experimental groups. CONCLUSION: Nanofibrous materials may prevent anastomotic leakage but delay healing.
Anastomotic leakage is a severe complication in gastrointestinal surgery. It is often a reason for reoperation together with intestinal passage blockage due to formation of peritoneal adhesions. Different materials as local prevention of these complications have been studied, none of which are nowadays routinely used in clinical practice. Nanofabrics created proved to promote healing with their structure similar to extracellular matrix. We decided to study their impact on anastomotic healing and formation of peritoneal adhesions. We performed an experiment on 24 piglets. We constructed 3 hand sutured end-to-end anastomoses on the small intestine of each pig. We covered the anastomoses with a sheet of polycaprolactone nanomaterial in the first experimental group, with a sheet of copolymer of polylactic acid with polycaprolactone in the second one and no fortifying material was used in the Control group. The animals were sacrificed after 3 weeks of observation. Clinical, biochemical and macroscopic signs of anastomotic leakage or intestinal obstruction were monitored, the quality of the scar tissue was assessed histologically, and a newly developed scoring system was employed to evaluate the presence of adhesions. The material is easy to manipulate with. There was no mortality or major morbidity in our groups. No statistical difference was found inbetween the groups in the matter of level of peritoneal adhesions or the quality of the anastomoses. We created a new adhesion scoring system. The material appears to be safe however needs to be studied further to prove its' positive effects.
- MeSH
- adheze tkání etiologie prevence a kontrola MeSH
- anastomóza chirurgická MeSH
- duodenum chirurgie MeSH
- hojení ran MeSH
- mikroskopie elektronová rastrovací MeSH
- modely nemocí na zvířatech MeSH
- náhodné rozdělení MeSH
- nanovlákna terapeutické užití ultrastruktura MeSH
- nemoci peritonea etiologie prevence a kontrola MeSH
- netěsnost anastomózy prevence a kontrola MeSH
- polyestery MeSH
- prasata MeSH
- testování materiálů MeSH
- tkáňové podpůrné struktury * MeSH
- zvířata MeSH
- Check Tag
- mužské pohlaví MeSH
- ženské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Závěrečná zpráva o řešení grantu Agentury pro zdravotnický výzkum MZ ČR
Nestr.
Materials that are currently used to fabricate vascular prostheses are non-degradable and thrombogenic. The aim of this project is to develop new tubular degradable scaffold made of nanofibers with three-dimensional double-layered structure. Biodegradable polymers will be electrospun to obtain nanofibrous vascular graft with desired properties. Inner layer will be made from thin fibers that will facilitate endothelial cell spreading from adjacent vessel. Outer layer will be composed of thicker fibers to enable smooth muscle cell infiltration into the 3D structure as in native vessel. These double-layered scaffolds will be tested mechanically to meet all requirements for vascular replacement in terms of tensile strength, elongation and sututre retention. The graft will be tested in vitro in static and dynamic conditions in bioreactor using endothelial and smooth muscle cells. The tissue remodeling process following the implantation will be predicted by macrophage polarization testing. Further in vivo tests will be carried out to investigate the patency of produced grafts.
Klinicky používané cévní náhrady jsou vyráběny z inertních materiálů a selhávají zejména díky trombogenicitě. Cílem tohoto projektuje je vyvinout nanovlákenný tkáňový nosič, který bude složen ze dvou vrstev. Biodegradabilní polymery budou zvlákněny metodou elektrostatického zvlákňování pro výrobu ideálního cévního bypassu. Vnitřní vrstva tohoto tkáňového nosiče bude tvořena tenkými nanovlákny, které budou usnadňovat endotelizaci vnitřního povrchu. Vnější vrstva bude vyrobena z vláken s větším průměrem, jejichž struktura bude umožňovat infiltraci hladkosvalových buněk dovnitř 3D struktury, jako je tomu u nativní cévy. Tyto dvouvrstevné cévní náhrady budou testovány mechanicky a poté optimalizovány tak, aby splňovaly nároky na pevnost v tahu, elasticitu a pevnost švu. Vyrobené cévní náhrady budou osázeny in vitro endotelovými a hladkosvalovými buňkami při statických i dynamických podmínkách v bioreaktoru. Proces remodelace tkáně, která následuje po implantaci do organismu, bude předpovídán sledováním polarizace makrofágů. Funkčnost náhrad bude testována in vivo.
- MeSH
- cévní protézy MeSH
- nanovlákna MeSH
- polymery MeSH
- testování materiálů MeSH
- vstřebatelné implantáty MeSH
- Konspekt
- Patologie. Klinická medicína
- NLK Obory
- technika lékařská, zdravotnický materiál a protetika
- NLK Publikační typ
- závěrečné zprávy o řešení grantu AZV MZ ČR
In the present work, we developed a novel needleless emulsion electrospinning technique that improves the production rate of the core/shell production process. The nanofibres are based on poly-ε-caprolactone (PCL) as a continuous phase combined with a droplet phase based on Pluronic F-68 (PF-68). The PCL-PF-68 nanofibres show a time-regulated release of active molecules. Needleless emulsion electrospinning was used to encapsulate a diverse set of compounds to the core phase [i.e. 5-(4,6-dichlorotriazinyl) aminofluorescein -PF-68, horseradish peroxidase, Tetramethylrhodamine-dextran, insulin growth factor-I, transforming growth factor-β and basic fibroblast growth factor]. In addition, the PF-68 facilitates the preservation of the bioactivity of delivered proteins. The system's potential was highlighted by an improvement in the metabolic activity and proliferation of mesenchymal stem cells. The developed system has the potential to deliver susceptible molecules in tissue-engineering applications.
- MeSH
- biokompatibilní materiály farmakologie MeSH
- dextrany chemie MeSH
- emulze chemie MeSH
- jehly MeSH
- kolagen typ II metabolismus MeSH
- křenová peroxidasa metabolismus MeSH
- mezenchymální kmenové buňky cytologie účinky léků metabolismus MeSH
- mezibuněčné signální peptidy a proteiny farmakologie MeSH
- miniaturní prasata MeSH
- nanovlákna chemie ultrastruktura MeSH
- poloxamer chemie MeSH
- polyestery chemie MeSH
- prasata MeSH
- proteiny aplikace a dávkování MeSH
- rhodaminy chemie MeSH
- tkáňové inženýrství metody MeSH
- tkáňové podpůrné struktury chemie MeSH
- zvířata MeSH
- Check Tag
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Additive manufacturing, also called 3D printing, is an effective method for preparing scaffolds with defined structure and porosity. The disadvantage of the technique is the excessive smoothness of the printed fibers, which does not support cell adhesion. In the present study, a 3D printed scaffold was combined with electrospun classic or structured nanofibers to promote cell adhesion. Structured nanofibers were used to improve the infiltration of cells into the scaffold. Electrospun layers were connected to 3D printed fibers by gluing, thus enabling the fabrication of scaffolds with unlimited thickness. The composite 3D printed/nanofibrous scaffolds were seeded with primary chondrocytes and tested in vitro for cell adhesion, proliferation and differentiation. The experiment showed excellent cell infiltration, viability, and good cell proliferation. On the other hand, partial chondrocyte dedifferentiation was shown. Other materials supporting chondrogenic differentiation will be investigated in future studies.
- MeSH
- 3D tisk * MeSH
- buněčná adheze fyziologie MeSH
- buněčná diferenciace fyziologie MeSH
- chondrocyty cytologie MeSH
- kultivované buňky fyziologie MeSH
- lidé MeSH
- nanovlákna * chemie MeSH
- proliferace buněk fyziologie MeSH
- tkáňové inženýrství metody MeSH
- tkáňové podpůrné struktury * MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- 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.
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
Fibrous scaffolds are desired in tissue engineering applications for their ability to mimic extracellular matrix. In this study we compared fibrous scaffolds prepared from polycaprolactone using three different fabrication methods, electrospinning (ES), electro-blowing and melt-blown combined with ES. Scaffolds differed in morphology, fiber diameters and pore sizes. Mesenchymal stem cell adhesion, proliferation and osteogenic differentiation on scaffolds was evaluated. The most promising scaffold was shown to be melt-blown in combination with ES which combined properties of both technologies. Microfibers enabled good cell infiltration and nanofibers enhanced cell adhesion. This scaffold was used for further testing in critical sized defects in rabbits. New bone tissue formation occurred from the side of the treated defects, compared to a control group where only fat tissue was present. Polycaprolactone fibrous scaffold prepared using a combination of melt-blown and ES technology seems to be promising for bone regeneration. The practical application of results is connected with enormous production capacity and low cost of materials produced by melt-blown technology, compared to other bone scaffold fabrication methods.
- MeSH
- buněčná adheze MeSH
- femur patologie MeSH
- kosti a kostní tkáň patologie MeSH
- králíci MeSH
- mezenchymální kmenové buňky cytologie MeSH
- mikroskopie elektronová rastrovací MeSH
- nanovlákna chemie MeSH
- osteogeneze účinky léků MeSH
- polymery chemie MeSH
- proliferace buněk MeSH
- regenerace kostí MeSH
- tkáňové inženýrství metody MeSH
- tkáňové podpůrné struktury chemie MeSH
- viabilita buněk MeSH
- zvířata MeSH
- Check Tag
- králíci MeSH
- mužské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
