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Alginate hydrogels are widely used in wound dressings due to hydrophilicity, biocompatibility, flexibility, and high sorption capacity, providing effective moisture regulation in wounds and inducing rapid granulation and reepithelization of the damaged tissue. However, these dressings are not bioactive so that different methodologies have been investigated to extend functionality of alginate hydrogels. In the present work, we show several approaches to achieve this aim by addition of different biologically active components. These include incorporation of silver nanoparticles as potent antimicrobial agents (1), bioactive honey components (2), activated charcoal (AC) particles as carriers of therapeutically active agents (3) as well as the use of Zn-alginate hydrogels that release zinc ions (4). The obtained composites were comprehensively characterized regarding composition, cytotoxicity, antibacterial activity, release kinetics of active agents and wound treatment in a rat model. Ag/alginate nanocomposite hydrogels releasing silver ions and/or nanoparticles exhibited high bactericidal activity against a broad spectrum of standard and multi-drug resistant clinical bacterial strains (Escherichia coli, Staphylococcus aureus, methicillin-resistant Staphylococcus aureus – MRSA, Acinetobacter baumannii and Pseudomonas aeruginosa). Especially interesting results were obtained against 13 clinical isolates of A. baumannii, which were completely extinguished over 48 h in 6 cases (2). However, in 3 clinical isolates, antibacterial effects were not noticed implying possibility for development of bacterial resistance to silver. In the treatment of 2nd degree burns in rats Ag/alginate nanocomposites exhibited the same efficiency as commercial medical products (5). Composite alginate hydrogels with immobilized AC particles impregnated with povidone iodine (PVP-I) as a model therapeutically active agent, were developed with the aim to provide controlled particle release in the wound without actually releasing the adsorbed substance, thus achieving the desired activity without adverse effects by systemic absorption. The composite Ca-alginate hydrogels induced strong bactericidal effects against two standard bacterial strains and clinical multi-resistant wound isolates (MRSA, E. coli, P. aeruginosa, Еnterococcus faecalis and Proteus mirabilis) without releasing PVP-I in the environment (3). Furthermore, composite Zn-alginate hydrogels released zinc ions in addition to AC particles with adsorbed PVP-I, which induced additional microbicidal effects on one wild yeast strain (Candida albicans). The obtained bactericidal effects were ascribed to effective adsorption of bacteria onto AC particles and further direct contact with the adsorbed iodine, while the antifungal activity against C. albicans was assigned to released Zn 2+. Overall, the developed composite alginate hydrogels have shown high potentials for utilization in variety of multifunctional wound dressings according to the specific needs. Alginatni hidrogelovi se široko koriste u oblogama za rane zbog svoje hidrofilnosti, biokompatibilnosti, fleksibilnosti i velikog sorpcionog kapaciteta čime obezbeđuju efikasnu regulaciju vlažnosti rane i podstiču brzu granulaciju i reepitelizaciju oštećenog tkiva. Međutim, ove obloge nisu bioaktivne tako da su istraživane različite metodologije kako bi se proširila funkcionalnost alginatnih hidrogelova. U ovom radu je prikazano nekoliko pristupa ostvarivanju tog cilja dodatkom različitih biološki aktivnih komponenata. Ovi pristupi uključuju inkorporaciju nanočestica srebra kao potentnog antimikrobnog agensa (1), bioaktivnih komponenata meda (2), čestica aktivnog uglja (AU) kao nosača terapeutski aktivnih agenasa (3), kao i primenu hidrogelova Zn-alginata koji otpuštaju jone cinka. Dobijeni kompoziti su sveobuhvatno karakterisani u pogledu sastava, citotoksičnosti, antibakterijske aktivnosti, kinetike otpuštanja aktivnih agenasa i tretmana rana u eksperimentalnom modelu opekotina na pacovima. Ag/alginatni nanokompozitni hydrogelovi su usled otpuštanja jona i/ili nanočestica srebra, pokazali izraženu baktericidnu aktivnost prema širokom spektru standardnih i kliničkih multi- rezistentnih bakterijskih sojeva (Escherichia coli, Staphylococcus aureus, meticilin-resistentni Staphylococcus aureus – MRSA, Acinetobacter baumannii i Pseudomonas aeruginosa). Posebno interesantni rezultati su dobijeni u kulturama 13 kliničkih izolata A. baumannii, gde je u 6 slučajeva postignut potpun baktericidan efekat u toku 48 h (2). Ipak, kod 3 klinička izolata nije postignuto antibakterijsko dejstvo što ukazuje na mogućnost razvoja bakterijske rezistencije na srebro. U tretmanu opekotina drugog stepena na pacovima, Ag/alginatni nanokompoziti su pokazali istu efikasnost kao komercijalni medicinski proizvodi. Kompozitni alginatni hidrogelovi sa imobilisanim česticama AU impregniranih povidon-jodom kao model terapeutski aktivnom komponentom, su razvijeni sa ciljem da obezbede kontrolisano otpuštanje čestica AU u rani bez otpuštanja adsorbovane supstance kako bi se na taj način postiglo željeno dejstvo bez neželjenih efekata sistemske apsorpcije. Kompozitni Ca-alginatni hidrogelovi su pokazali jake baktericidne efekte na dva standardna bakterijska soja i nekoliko kliničkih multi- rezistentnih izolata iz rana (MRSA, E. coli, P. aeruginosa, Еnterococcus faecalis i Proteus mirabilis) bez otpuštanja povidon-joda u okolinu (3). Isto tako, kompozitni Zn-alginatni hidrogelovi su otpuštali jone cinka uz otpuštanje AU čestica sa adsorbovanim povidon-jodom što je prouzrokovalo dodatno mikrobicidno dejstvo na jedan divlji soj gljivice Candida albicans. Dobijeni baktericidni efekti su pripisani efikasnoj adsorpciji bakterija na čestice AU i daljem direktnom kontaktu adsorbovanog joda sa ćelijskom membranom bakterija, dok je antifungalna aktivnost u odnosu na C. albicans pripisana otpuštenim Zn 2+ jonima. Može se zaključiti da su razvijeni kompozitni alginatni hdrogelovi pokazali veliki potencijal za primenu u raznovrsnim multifunkcionalnim oblogama za rane prilagođenim specifičnim potrebama. Drugi naučni simpozijum Saveza farmaceutskih udruženja Srbije sa međunarodnim učešćem, 28. 10. 2021. Beograd

Today, the development of new composite biomaterials for wound treatment application is based on knowledge of different factors which affects the process of wound healing in order to identify solutions for specific problems related to chronic wounds. The aim of this thesis was to develop new composite hydrogels based on alginate and activated charcoal particles (AC) which would show beneficial effects in potential wound treatment application. Specifically, the aim was to develop composites which would during contact with biological fluids, release AC particles in a controlled and efficient manner during a prolonged period of time. Simultaneously, AC particles could be carriers of different active agents while alginate hydrogel would efficiently absorb the fluids. Povidone-iodine (PVP-I) and lignin model compounds (LMC) were chosen as model substances of active agents with antimicrobial activity. The results showed that the composite Ca-alginate beads (CaA/AC) were successfully obtained by extrusion method using mixtures of alginate in concentration range of 0.5-2 % w/w and AC in a concentration range of 1-20 % w/w. The diffusion model was successfully applied on the release kinetics experimental data of all groups of A/AC beads, except in the case of the beads obtained from the mixture with the optimal composition. In this case, the release of AC particles was a result of diffusion as well as erosion and the Korsmeyer-Peppas model was successfully applied. Zn0.5/H composite beads were successfully obtained by extrusion of optimal mixture in the gelling bath that contained Zn ions. The beads based on Ca- and Znalginate obtained from the optimal mixture (Ca- and Zn0.5/H beads) released around 50-60 % of iv the total AC content during a 5 day period, which is a period relevant for chronic wound treatment. These beads were successfully impregnated with the first model substance (PVP-I). The desorption studies established that PVP-I was firmly adsorbed onto AC particles and that the level of desorption into alginate matrix as well as saline solution was below the detection level. The second model substance (LMC) was successfully absorbed and the level of desorption in saline solution was below the detection level as well. Both composite Ca- and Zn-alginate beads impregnated with PVP-I showed high antimicrobial activity in the suspensions of all examined ATCC and clinical strains as well. The antimicrobial effect of adsorbed iodine can be explained by direct contact with the cell membrane of microorganisms which was enough to establish bactericidal effect in case of Gram positive and Gram negative bacteria while the yeast Candida albicans was the most resistant. On the contrary, AC particles with adsorbed LMC did not exhibit antimicrobial effect in any of the examined suspensions which demands a further investigation of adsorption mechanism and antimicrobial activity of LMC as well. Composite Ca- and Zn-alginate hydrogels with incorporated AC particles with adsorbed PVP-I showed potential for wound dressing application because of its high efficiency, availability and broth antimicrobial spectrum of iodine as well as its potential synergistic effect with released cations that exhibit wound healing effect. Danas, razvoj novih kompozitnih biomaterijala za medicinsku primenu u terapiji rana bazira se na poznavanju različitih faktora povezanih sa procesom zarastanja i podrazumeva rešavanje specifičnih problema koji prate hronične rane. Cilj ove doktorske disertacije bio je razvoj novih kompozitnih hidrogelova na bazi alginata i čestica aktivnog uglja (AU) koji će u potencijalnoj primeni u tretmanu rana ispoljiti više povoljnih efekata. Naime, cilj je bio razvoj kompozita iz koga se, u dodiru sa biološkim fluidima, čestice AU kontrolisano i efikasno otpuštaju tokom dužeg perioda. Pri tome, čestice AU mogu biti nosači različitih aktivnih agenasa, dok bi alginatni hidrogel efikasno upijao vlagu. Kao model aktivne supstance izabrani su povidon-jod (PVP-I) i lignin model jedinjenja (LMJ) kao agensi koji ispoljavaju antimikrobno dejstvo. Rezultati istraživanje su pokazali da su metodom ekstruzije uspešno dobijene kompozitne Ca-alginatne čestice (CaA/AU) i to iz smeša alginata koncentracija u opsegu od 0,5 - 2 mas. % i AU u opsegu od 1-20 mas. %. Ispitivanjem kinetike otpuštanja AU iz suvih CaA/AU čestica u fiziološkom rastvoru utvrđeno je da je sastav optimalne smeše 0,5 mas. % alginata i 20 mas. % AU. Kinetika otpuštanja AU je uspešno opisana modelom unutrašnje difuzije osim u slučaju čestica dobijenih iz smeše optimalnog sastava gde je otpuštanje uzrokovano i erozijom čestica što je opisano Korsmejer-Pepasovim modelom Ekstruzijom smeše optimalnog sastava u rastvor za geliranje koji je sadržao jone cinka uspešno su dobjene i Zn0,5/V kompozitne čestice. Čestice na bazi Ca- i Zn-alginata dobijene iz smeše optimalnog sastava (Ca- i Zn0,5/V čestice) su u ii fiziološkom rastvoru otpustile oko 50 - 60 % ukupnog sadržaja AU tokom perioda od 5 dana koji je relevantan za tretman hroničnih rana. Ove čestice su zatim uspešno impregnirane prvom model supstancom (PVP-I), a u ispitivanjima desorpcije utvrđeno je da je PVP-I čvrsto vezan za čestice AU i da je stepen desorpcije u okolni alginatni matriks, kao i u fiziološki rastvor zanemarljiv. Na čestice AU je takođe uspešno adsorbovana i druga model supstanca (LMJ) i pokazano je da ne dolazi ni u ovom slučaju ne dolazi do desorpcije u fiziološkom rastvoru. Kompozitne Ca- i Zn-alginatne čestice impregnirane sa PVP-I su pokazale izrazitu antimikrobnu aktivnost u suspenzijama svih ispitivanih, standardnih i kliničkih sojeva mikroorganizama. Antimikrobni efekat adsorbovanog joda se može pripisati direktnom kontaktu sa ćelijskom membranom mikroorganizama što je u slučaju ispitivanih Gram pozitivnih i Gram negativnih bakterija bilo dovoljno za baktericidni efekat, za razliku od gljivice C. albicans koja se pokazala najotpornijom. Sa druge strane čestice AU sa adsorbovanim LMJ nisu pokazale antimikrobne efekte u ispitivanim suspenzijama što zahteva dalja istraživanja mehanizma adsorpcije ovih jedinjenja, kao i mehanizma njihovog antimikrobnog dejstva. Kompozitni Ca- i Zn-alginatni hidrogelovi sa sadržanim česticama AU na kojima je adsorbovan PVP-I su pokazali potencijal za primenu u oblogama za rane zbog efikasnosti, dostupnosti i širokog spektra dejstva joda, kao i potencijalnog sinergističkog dejstva sa otpuštenim katjonima koji imaju pozitivan efekat na zarastanje oštećenog tkiva.

Efficient transport of regulatory molecules is one of the main requirements for directing the growth and differentiation of bone marrow stromal cells (BMSC). We have designed and utilized a packed bed bioreactor system for cultivation of murine BMSC immobilized in alginate micro beads produced by electrostatic droplet generation. Continuous medium perfusion at velocities that are physiological for cartilage and bone (~ 100 m/s) provided convective mass transport through the packed bed while the small bead diameter (~ 500µm) ensured short diffusion distances to the immobilized cells. Over 5 weeks of cultivation, the cells remained viable at a constant density whereas the alginate micro beads retained size and spherical shape. Cell density used in this study ( 5 x 106 cells/ml) was found to be too low to result in cartilage tissue formation. However, in several cases, loosely bonded groups of beads and merged beads without visible boundaries were observed, implying that higher cell densities may lead to development of a continuous extra cellular matrix. This study indicates the potentials of the packed bed bioreactor system in conjunction with alginate micro beads as cell carriers, for precise regulation of the cellular microenvironment in cartilage tissue engineering. Efikasan prenos regulatornih molekula je jedan od osnovnih zahteva u kulturama ćelija kostne srži radi podsticaja ćelija na diferencijaciju u željenom pravcu. U ovom radu je razvijen i primenjen bioreaktorski sistem sa pakovanim slojem za kultivaciju ćelija kostne srži miša imobilisanih u alginatne mikročestice proizvedene tehnikom elektrostatičke ekstruzije. Kontinualnim protokom medijuma pri fiziološkim brzinama strujanja (~ 100 µm/s) obezbeđen je konvektivni prenos mase u pakovanom sloju dok su malim prečnikom čestica (~ 500µm) ostvarena mala rastojanja za difuzioni prenos mase do imobilisanih ćelija. U toku 5 nedelja kultivacije, ćelije su ostale vijabilne pri konstantnoj ćelijskoj gustini dok su alginatne mikročestice zadržale veličinu i sferičan oblik. Pokazalo se da je koncentracija ćelija korišćena u ovom radu (5 x 106 cells/ml) suviše niska za formiranje tkiva hrskavice. Međutim, u nekoliko slučajeva nađene su slabo vezane grupe mikročestica, a ponegde i potpuno stopljene mikročestica bez primetnih granica što navodi na pretpostavku da bi pri višim koncentracijama ćelija bio moguć razvoj kontinualnog ekstracelularnog matriksa. Rezultati dobijeni u ovom radu ukazuju na mogućnost primene bioreaktorskog sistema sa pakovanim slojem alginatnih mikročestica, nosača ćelija, za preciznu regulaciju mikro-okoline ćelija u inženjerstvu tkiva hrskavice.