Hydrogel Containing Anti-CD44-Labeled Microparticles, Guide Bone Tissue Formation in Osteochondral Defects in Rabbits

Hydrogels are suitable for osteochondral defect regeneration as they mimic the viscoelastic environment of cartilage. However, their biomechanical properties are not sufficient to withstand high mechanical forces. Therefore, we have prepared electrospun poly-ε-caprolactone-chitosan (PCL-chit) and poly(ethylene oxide)-chitosan (PEO-chit) nanofibers, and FTIR analysis confirmed successful blending of chitosan with other polymers. The biocompatibility of PCL-chit and PEO-chit scaffolds was tested; fibrochondrocytes and chondrocytes seeded on PCL-chit showed superior metabolic activity. The PCL-chit nanofibers were cryogenically grinded into microparticles (mean size of about 500 µm) and further modified by polyethylene glycol–biotin in order to bind the anti-CD44 antibody, a glycoprotein interacting with hyaluronic acid (PCL-chit-PEGb-antiCD44). The PCL-chit or PCL-chit-PEGb-antiCD44 microparticles were mixed with a composite gel (collagen/fibrin/platelet rich plasma) to improve its biomechanical properties. The storage modulus was higher in the composite gel with microparticles compared to fibrin. The Eloss of the composite gel and fibrin was higher than that of the composite gel with microparticles. The composite gel either with or without microparticles was further tested in vivo in a model of osteochondral defects in rabbits. PCL-chit-PEGb-antiCD44 significantly enhanced osteogenic regeneration, mainly by desmogenous ossification, but decreased chondrogenic differentiation in the defects. PCL-chit-PEGb showed a more homogeneous distribution of hyaline cartilage and enhanced hyaline cartilage differentiation

Histological evaluation of experimental models of liver diseases

Animal models are widely used for research of liver diseases pathogenesis and progression and for development of new treatment strategies in hepatology. The dissertation thesis focuses on large animal model, specifically swine. The use of animals, which are anatomically and physiologically close to humans, allows us to bridge the gap between the experimental and human medicine. Histopathological analysis of the liver biopsies is still a fundamental part of liver disease diagnosis and therefore, it is also a part of the experimental design of the studies using the porcine liver. Our aim was to apply qualitative and quantitative histological methods of evaluation on porcine liver and to assess their usability in experimental medicine. The quantitative methods included automated image analysis as well as stereological methods, which guaranteed high reproducibility and comparability of the experimental results. The dissertation thesis is based on 10 manuscripts. Three of them are published reviews associated with the main topic of the thesis. Seven original manuscripts resulted from six experimental studies - their six conclusions are listed bellow: Conclusion 1: We developed an open-source software QuantAn for quantification of microvessels visualized by 3D imaging methods, such as computed tomography...Zvířecí modely jsou hojně využívány při studiu patogeneze a progrese jaterních onemocnění či při vývoji nových strategií jejich terapeutického ovlivnění. Zaměření této dizertační práce na velký zvířecí model, prase domácí, odráží současný trend na poli experimentální medicíny, tedy využití modelů co nejbližších člověku tak, aby byly výsledky experimentů co nejpřesněji přenositelné do humánní medicíny. V rutinní patologické praxi je histologické hodnocení nedílnou součástí procesu stanovení diagnózy a závažnosti jaterního poškození. Analogicky je tedy histopatologické vyšetření tkáně zařazeno do designu experimentálních studií na játrech prasete. Naším cílem bylo aplikovat metody kvalitativního a kvantitativního histologického hodnocení a ověřit jejich využitelnost na játrech prasete. Metody kvantifikace zahrnovaly jak automatickou obrazovou analýzu dat, tak stereologické metody zaručující vysokou reprodukovatelnost studií a porovnatelnost výsledků z různých experimentů. Dizertační práce je založena na celkem 10 pracích: třech přehledových pracích a sedmi výsledkových pracích. Výsledkové práce vycházejí ze šesti studií, jejichž šest závěrů můžeme shrnout následovně: Závěr 1: Vyvinuli jsme open-source software QuantAn pro automatickou analýzu mikrocév nasnímaných pomocí 3D zobrazovacích metod, např....Ústav histologie a embryologieLékařská fakulta v PlzniFaculty of Medicine in Pilse

Histological evaluation of experimental models of liver diseases

Animal models are widely used for research of liver diseases pathogenesis and progression and for development of new treatment strategies in hepatology. The dissertation thesis focuses on large animal model, specifically swine. The use of animals, which are anatomically and physiologically close to humans, allows us to bridge the gap between the experimental and human medicine. Histopathological analysis of the liver biopsies is still a fundamental part of liver disease diagnosis and therefore, it is also a part of the experimental design of the studies using the porcine liver. Our aim was to apply qualitative and quantitative histological methods of evaluation on porcine liver and to assess their usability in experimental medicine. The quantitative methods included automated image analysis as well as stereological methods, which guaranteed high reproducibility and comparability of the experimental results. The dissertation thesis is based on 10 manuscripts. Three of them are published reviews associated with the main topic of the thesis. Seven original manuscripts resulted from six experimental studies - their six conclusions are listed bellow: Conclusion 1: We developed an open-source software QuantAn for quantification of microvessels visualized by 3D imaging methods, such as computed tomography..

Animal models of liver diseases and their application in experimental surgery

Akutní a chronická jaterní onemocnění představují širokou skupinu chorob, které často ohrožují pacienty na životě. Pochopení mechanismů stojících za patogenezí a progresí jaterního poškození je klíčové pro vývoj nových terapeutických strategií a léků. Nejvýznamnějším faktorem limitujícím studium patogeneze a progrese jaterních onemocnění je nedostatek vhodných zvířecích modelů. Do současné doby bylo etablováno množství zvířecích modelů napodobujících jaterní onemocnění u lidí. Toto přehledné sdělení shrnuje dosud publikované zvířecí modely chronických i akutních jaterních onemocnění se zvláštním důrazem na velké zvířecí modely a jejich využití v experimentální chirurgii.Both acute and chronic liver diseases are frequent and potentially lethal conditions. Development of new therapeutic strategies and drugs depends on understanding of liver injury pathogenesis and progression, which can be studied on suitable animal models. Due to the complexity of liver injury, the understanding of underlying mechanisms of liver diseases and their treatment has been limited by the lack of satisfactory animal models. Although significant progress has been made, the research in hepatology should continue to establish animal models anatomically and physiologically as close to human as possible to allow for translation of the experimental results to human medicine. This review presents various approaches to the study of acute and chronic liver diseases in animal models, with special emphasis on large animal models and their role in experimental surgery

Hydrogel Containing Anti-CD44-Labeled Microparticles, Guide Bone Tissue Formation in Osteochondral Defects in Rabbits

Hydrogels are suitable for osteochondral defect regeneration as they mimic the viscoelastic environment of cartilage. However, their biomechanical properties are not sufficient to withstand high mechanical forces. Therefore, we have prepared electrospun poly-ε-caprolactone-chitosan (PCL-chit) and poly(ethylene oxide)-chitosan (PEO-chit) nanofibers, and FTIR analysis confirmed successful blending of chitosan with other polymers. The biocompatibility of PCL-chit and PEO-chit scaffolds was tested; fibrochondrocytes and chondrocytes seeded on PCL-chit showed superior metabolic activity. The PCL-chit nanofibers were cryogenically grinded into microparticles (mean size of about 500 µm) and further modified by polyethylene glycol–biotin in order to bind the anti-CD44 antibody, a glycoprotein interacting with hyaluronic acid (PCL-chit-PEGb-antiCD44). The PCL-chit or PCL-chit-PEGb-antiCD44 microparticles were mixed with a composite gel (collagen/fibrin/platelet rich plasma) to improve its biomechanical properties. The storage modulus was higher in the composite gel with microparticles compared to fibrin. The Eloss of the composite gel and fibrin was higher than that of the composite gel with microparticles. The composite gel either with or without microparticles was further tested in vivo in a model of osteochondral defects in rabbits. PCL-chit-PEGb-antiCD44 significantly enhanced osteogenic regeneration, mainly by desmogenous ossification, but decreased chondrogenic differentiation in the defects. PCL-chit-PEGb showed a more homogeneous distribution of hyaline cartilage and enhanced hyaline cartilage differentiation