Vývoj a verifikace in vitro modelu kosti pro testování polymerních tkáňových nosičů
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2025-10-22T00:00:00Z
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Tato práce pojednává o vývoji in vitro modelu kosti pro detailní testování kostních náhrad, aby se předešlo nadměrnému testování na zvířatech. Buněčné monokultury používané pro in vitro testování kostních náhrad postrádají komplexnost celé tkáně či organismu, takže získané výsledky mají omezenou vypovídací hodnotu. Ko-kultury různých typů kostních buněk umožňují přirozenou signalizaci a komunikaci, což lépe napodobuje hojení v in vivo prostředí. Hlavními aktéry hojení jsou osteoklasty (OCs), degradující poškozenou tkáň, a osteoblasty (OBs), tvořící novou tkáň. Tyto dva buněčné typy byly použity pro vývoj in vitro modelu kosti. Buňky byly izolovány z potkanů, byl potvrzen jejich fenotyp a byly optimalizovány podmínky ko-kultury, tj. kultivační médium, poměr buněčných typů při nasazení a použité suplementy do média. Funkčnost in vitro modelu byla ověřena pomocí nanovláken z poly--kaprolaktonu (PCL) obsahujících 0, 10 a 20 % w/v hydroxyapatitu (HAp) elektrostaticky zvlákněných ve střídavém (AC) nebo stejnosměrném (DC) proudu, a chování ko-kultury bylo porovnáno s rutinně používanou monokulturou OBs. PCL bez HAp elektrostaticky zvlákněný v DC vykazoval ve srovnání s ostatními nanovlákennými materiály menší medián průměru vláken, úzký rozsah hustoty pravděpodobnosti poloměru pórů a malý kontaktní úhel. Tyto parametry patrně vedly k lepší adhezi buněk, osteoklastogenezi a osteogenezi. Také byly zaznamenány rozdíly v chování buněk mezi ko-kulturou a monokulturou OBs. Kostní ko-kultura byla dále použita k testování účinku nanovláken uvolňujících Alendronát (ALN), lék na osteoporózu, určený k lokální léčbě osteoporózy v místě zlomeniny. Byla hledána koncentrace ALN, která není cytotoxická, ale inhibuje osteoklastogenezi a poté byla vytvořena nanovlákna s ALN nebo v kombinaci s HAp, který uvolňování ALN zpomalil. Byly připraveny ko-kultury z osteoporotických a zdravých (kontrolních) potkanů, avšak nebyl pozorován žádný zjevný rozdíl v chování buněk v reakci na nanovlákna uvolňující ALN různou rychlostí. Pozorovali jsme však nižší metabolickou aktivitu a proliferaci a vyšší osteogenní a osteoklastogenní differenciaci ko-kultur z osteoporotických potkanů ve srovnání s ko-kulturami ze zdravých potkanů. Závěrem lze říci, že byl vyvinut in vitro model kosti z ko-kultury OCs a OBs a rozdíl v chování buněk ve srovnání s monokulturou OBs ukázal důležitost jeho použití pro in vitro testování kostních náhrad.
This thesis deals with developing an in vitro bone model for detailed testing of bone substitutes to avoid excessive animal testing. The cell monocultures used lack the complexity of a whole tissue or an organism, so the results obtained are of limited information. Co-cultures of bone cell types enable natural signaling and communication that better mimic the healing in the in vivo environment. The main players in bone healing are osteoclasts (OCs) degrading the damaged tissue, and osteoblasts (OBs) forming new tissue. These two cell types were used to develop the in vitro bone model. Cells were isolated from rats, their phenotype was confirmed, and the co-culture conditions, i.e., culture medium, the seeding ratio between the cell types, and supplements used, were optimized. The functionality of the in vitro bone model for testing bone substitutes was verified with poly--caprolactone (PCL) nanofibers containing 0, 10, and 20% w/v hydroxyapatite (HAp) electrospun by alternating current (AC) or direct current (DC) methods and compared with the routinely used OBs monoculture. PCL without HAp electrospun by DC showed smaller median fiber diameter, a narrow range of probability density of pore radius, and a low contact angle compared to other nanofibers. These parameters probably led to better cell adhesion, osteoclastogenesis, and osteogenic differentiation compared to other nanofibrous materials. Differences in cell behavior were observed between the co-culture and the OBs monoculture. The bone co-culture was also used to test the effect of nanofibers releasing Alendronate (ALN), an osteoporosis drug, designed to treat osteoporosis locally at the fracture site. A concentration of ALN that is not cytotoxic but inhibits osteoclastogenesis was sought. Nanofibers were prepared with ALN alone or in combination with HAp, which slows ALN release. No apparent difference was observed between the nanofibers with different ALN release kinetics on co-cultures from osteoporotic and healthy (control) rats. However, we observed lower metabolic activity and proliferation and higher osteogenic and osteoclastogenic differentiation in co-cultures from osteoporotic rats compared to healthy rats. In conclusion, the in vitro bone model consisting of a co-culture of OCs and OBs was developed, and the differences in cell behavior compared to OBs monoculture showed the importance of its use for in vitro testing of bone substitutes.
This thesis deals with developing an in vitro bone model for detailed testing of bone substitutes to avoid excessive animal testing. The cell monocultures used lack the complexity of a whole tissue or an organism, so the results obtained are of limited information. Co-cultures of bone cell types enable natural signaling and communication that better mimic the healing in the in vivo environment. The main players in bone healing are osteoclasts (OCs) degrading the damaged tissue, and osteoblasts (OBs) forming new tissue. These two cell types were used to develop the in vitro bone model. Cells were isolated from rats, their phenotype was confirmed, and the co-culture conditions, i.e., culture medium, the seeding ratio between the cell types, and supplements used, were optimized. The functionality of the in vitro bone model for testing bone substitutes was verified with poly--caprolactone (PCL) nanofibers containing 0, 10, and 20% w/v hydroxyapatite (HAp) electrospun by alternating current (AC) or direct current (DC) methods and compared with the routinely used OBs monoculture. PCL without HAp electrospun by DC showed smaller median fiber diameter, a narrow range of probability density of pore radius, and a low contact angle compared to other nanofibers. These parameters probably led to better cell adhesion, osteoclastogenesis, and osteogenic differentiation compared to other nanofibrous materials. Differences in cell behavior were observed between the co-culture and the OBs monoculture. The bone co-culture was also used to test the effect of nanofibers releasing Alendronate (ALN), an osteoporosis drug, designed to treat osteoporosis locally at the fracture site. A concentration of ALN that is not cytotoxic but inhibits osteoclastogenesis was sought. Nanofibers were prepared with ALN alone or in combination with HAp, which slows ALN release. No apparent difference was observed between the nanofibers with different ALN release kinetics on co-cultures from osteoporotic and healthy (control) rats. However, we observed lower metabolic activity and proliferation and higher osteogenic and osteoclastogenic differentiation in co-cultures from osteoporotic rats compared to healthy rats. In conclusion, the in vitro bone model consisting of a co-culture of OCs and OBs was developed, and the differences in cell behavior compared to OBs monoculture showed the importance of its use for in vitro testing of bone substitutes.
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Kost, ko-kultura, osteoblast, osteoklast, nanovlákna, poly--kaprolakton, hydroxyapatit, osteoporóza.