Izolacija in funkcijsko vrednotenje rastnih dejavnikov iz adherentnih krvnih celic in vitro ter vzporedni razvoj nanovlaken za celjenje ran

Vogrinčič, Tjaša (author), Jeras, Matjaž (mentor), Kocbek, Petra (comentor)

Rana je definirana kot poškodba kože, povzročena z zunanjimi dejavniki, kirurškim posegom ali patološkimi stanji (diabetes, žilne bolezni). Če celjenje rane traja dlje kot 8 tednov, govorimo o kroničnih ranah, ki pomembno vplivajo na kakovost življenja obolelega in predstavljajo vedno večji zdravstveno-ekonomski družbeni problem. V kroničnih ranah, v primerjavi z akutnimi, zasledimo nižje koncentracije rastnih dejavnikov, kar nakazuje, da je njihovo pomankanje lahko ključni dejavnik tveganja za nastanek kroničnega stanja. Namen magistrske naloge je bil izolirati rastne dejavnike iz adherentnih mononuklearnih krvnih celic, izoliranih iz periferne krvi, za potencialno uporabo pri celjenju ran. Mononuklearne krvne celice smo izolirali iz filtrov za filtriranje krvi krvodajalcev, nato pa na dva načina, in sicer s pritrjevanjem na plastično podlago in z uporabo kolone z najlonskimi vlakni, iz njih pridobili adherentne celice. Slednje smo hipoosmotsko aktivirali in nato ovrednotili vpliv sproščenih citokinov oz. rastnih dejavnikov na proliferacijo človeških keratinocitov in vitro. Dokazali smo, da nam je uspelo pridobiti rastne dejavnike, ki pomembno vplivajo na rast in razmnoževanje keratinocitov, s čimer pospešujejo celjenje ran. Z določanjem količine rastnih dejavnikov s pretočno citometrijo pa nam je uspelo dokazati, da je bilo pridobivanje rastnih dejavnikov uspešnejše takrat, ko smo za adhezijo mononuklearnih celic uporabili kolono z najlonskimi vlakni. Ker so citokini oz. rastni dejavniki proteini in je njihova stabilnost in vivo omejena, so za njihov učinkovit vnos v telo ključni ustrezni dostavni sistemi. Ker smo želeli izdelati oblogo za rane z rastnimi dejavniki, smo kot dostavni sistem izbrali nanovlakna. V ta namen smo z metodo elektrostatskega sukanja skušali izdelati hidrofilna nanovlakna iz hitosana ali alginata z dodatkom polietilenoksida. Nanovlaken iz hitosana in polietilenoksida nismo uspeli izdelati. Uspešni pa smo bili pri pripravi nanovlaken iz alginata in polietilenoksida. Z optimizacijo procesnih parametrov smo izdelali gladka nanovlakna brez vozlov. Nato smo preučevali njihovo obstojnost v vodi in ugotovili, da nanovlakna iz alginata in polietilenoksida v prečiščeni vodi niso obstojna, saj se takoj raztopijo. Zato smo jih ionsko premrežili z raztopino CaCl2. Premrežena nanovlakna so v prečiščeni vodi ohranila vlaknato strukturo vsaj 72 h, kar kaže, da bi bila primerna za potencialno izdelavo oblog za rane z vgrajenimi rastnimi dejavniki.

• Language: Slovenian
• Keywords: celjenje ran, elektrostatsko sukanje, hidrofilna nanovlakna, obloge za rane, rastni dejavniki
• Work type: Master's thesis/paper
• Organization: FFA - Faculty of Pharmacy
• Year: 2024
• PID: 20.500.12556/RUL-156399
• Publication date in RUL: 24.05.2024

VOGRINČIČ, Tjaša, 2024, Izolacija in funkcijsko vrednotenje rastnih dejavnikov iz adherentnih krvnih celic in vitro ter vzporedni razvoj nanovlaken za celjenje ran [online]. Master’s thesis. [Accessed 11 April 2025]. Retrieved from: https://repozitorij.uni-lj.si/IzpisGradiva.php?lang=eng&id=156399

Secondary language

• Language: English
• Title: Isolation and in vitro functional evaluation of growth factors from adherent blood cells, and a paralel development of nanofibres for wound healing

Abstract

Wounds are defined as skin damage caused by external factors, surgery or pathological conditions (diabetes, vascular diseases). If wound healing lasts longer than 8 weeks, we are talking about chronic wounds, which significantly impair the quality of life of those affected and represent an increasing health and economic social problem. Lower concentrations of growth factors are observed in chronic wounds compared to acute wounds, suggesting that their deficiency could be a major risk factor for the development of chronic condition. The aim of the master's thesis was to isolate growth factors from adherent mononuclear blood cells, isolated from peripheral blood in order to potentially utilize them for wound healing. Mononuclear blood cells were isolated from donor blood filters by attaching them to a plastic substrate and using a nylon fibre column, to extract adherent blood cells. The latter were hypoosmotically activated and then the influence of released cytokines or growth factors on the proliferation of human keratinocytes was studied in vitro. We have shown that we have succeeded in obtaining growth factors that significantly influence the growth and proliferation of keratinocytes and thus accelerate wound healing. By determining the amount of growth factors using flow cytometry, we were able to demonstrate that the extraction of growth factors was more successful when we used a nylon fibre column to adhere mononuclear cells. Since cytokines or growth factors are proteins and their stability in vivo is limited, appropriate delivery systems are crucial for their effective uptake into the body. Since we wanted to produce a wound dressing with growth factors, we chose nanofibers as a delivery system. For this purpose, using the electrostatic twisting method, we tried to produce hydrophilic nanofibers from chitosan or alginate, with the addition of polyethylene oxide. We did not succeed in producing chitosan and polyethylene oxide nanofibers. However, we were successful in producing nanofibers from alginate and polyethylene oxide. By optimizing the process parameters, we produced smooth nanofibers, without knots. We then investigated their stability in water and found that nanofibers made of alginate and polyethylene oxide are not stable in purified water, as they dissolve immediately. Therefore, they were ionically crosslinked with a CaCl2 solution. The cross-linked nanofibers retained their fibrous structure in purified water for at least 72 h, indicating that they would be suitable for the potential production of wound dressings with incorporated growth factors.

• Keywords: wound healing, electrostatic twisting, hydrophilic nanofibers, wound dressings, growth factors