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Cijeljenje rane je kompleksan proces koji uključuje interakcije različitih tipova stanica, izvanstaničnog matriksa i različitih faktora rasta. U procesu cijeljenja rane važnu ulogu imaju fibroblasti koji tijekom inflamatorne faze kemotaksijom migriraju u područje rane gdje sintetiziraju i kasnije remodeliraju novi izvanstanični matriks. S razvojem terapijskih sustava s potencijalnim učinkom na cijeljenje rane, raste i potreba za razvijanjem in vitro modela rane koji mogu poslužiti kao zamjena za in vivo modele pri procjeni potencijala razvijenih sustava. In vitro modeli omogućuju jeftino i brzo probiranje tvari, materijala i terapijskih sustava koji bi se mogli potencijalno koristiti u daljnjim ispitivanjima. Time se smanjuje broj ispitivanja provedenih u laboratorijskih životinja. Cilj ovog rada bio je ispitati mogućnost korištenja MJ90 fibroblasta kože u pripravi in vitro modela rane koji bi omogućio procjenu učinka lecitinsko-kitozanskih nanočestica s melatoninom na proliferaciju i migraciju fibroblasta in vitro. Razvoj diskriminatornog modela omogućio bi probir formulacija s najvećim potencijalnim učinkom na cijeljenje rane.Lecitinsko-kitozanske nanočestice s melatoninom uspješno su pripravljene ionskom interakcijom lecitina i kitozana. Korišten je kitozan molekulske mase 50-150 kDa i stupnja deacetilacije .90%. Pripravljene su nanočestice srednjeg promjera od 254,6 ± 5,0 nm, pozitivnog zeta-potencijala od 24,2 ± 0,8 mV te zadovoljavajućeg sadržaja melatonina od 3,8 ± 0,4 %. Variranjem eksperimentalnih uvjeta (obloženost jažica fibronektinom, koncentracija FBS-a u hranidbenom mediju nakon postizanja konfluentnosti) razvijena su četiri protokola priprave modelne rane temeljene na imortaliziranoj staničnoj liniji MJ90 fibroblasta. Pri uvjetima koji su isključivali oblaganje jažica fibronektinom, te uključivali zamjenu hranidbenog medija s 10% FBS-a hranidbenim medijem s 0,5 % FBS-a nakon postizanja konfluentnosti, pripravljene su modelne rane zadovoljavajuće diskriminatornosti te je omogućena procjena učinka ispitivanih sustava na brzinu cijeljenja in vitro. Primjenom razvijenog modela rane, utvrđen je pozitivan učinak suspenzije lecitinsko-kitozanskih nanočestica s melatoninom na proliferaciju i migraciju fibroblasta in vitro. Wound healing is a complex process which includes interactions of different cell types, extracellular matrix and different growth factors. In the process of wound healing fibroblasts have an important role. During the inflammatory phase they migrate by chemotaxis to the wound site where they synthesize and later remodel extracellular matrix. Development of drug delivery systems with the potential effect on wound healing increases the need for the development of in vitro wound healing models which can serve as a substitute for in vivo wound models in evaluation of the potential of systems developed. In vitro models allow for cheap and fast screening of substances, materials and drug delivery systems which could be potentially used for further evaluations. In that way the number of tests performed on laboratory animals can be reduced. The aim of this work was to investigate the possibility of using MJ90 dermal fibroblasts in the preparation of in vitro wound healing model which would enable the evaluation of the effect of melatonin-loaded lecithin/chitosan nanoparticles on the proliferation and migration of fibroblasts in vitro. Development of the discriminative model would allow for screening of the formulations with the greatest potential effect on the wound healing. Melatonin-loaded lecithin/chitosan nanoparticles were successfully prepared by ionic interaction between lecithin and chitosan. Chitosan used was characterised by the molecular weight of 50-150 kDa and deacetylation degree above 90 %. Nanoparticles had mean diameter of 254.6 ± 5.0 nm, positive zeta-potential of 24.2 ± 0.8 mV and sufficient melatonin entrapment (3.8 ± 0.4 %). By varying experimental conditions (fibronectin plate coating, concentration of FBS in the medium after reaching the confluence) four protocols for the preparation of wound healing model based on immortalized MJ90 fibroblast cell line were developed. Under conditions excluding fibronectin plate coating but including replacement of medium supplemented with 10 % of FBS with medium supplemented with 0.5 % FBS after reaching the confluence, discriminative wound healing model was prepared, allowing for the evaluation of the effect of the tested drug delivery systems on the wound healing in vitro. Using the developed wound healing model, the positive effect of melatonin-loaded lecithin/chitosan nanoparticles on the proliferation and migration of fibroblasts was observed. |
