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The fetal membranes provide a supportive environment for the growing embryo and later fetus. Due to their versatile properties, the use of fetal membranes in tissue engineering and regenerative medicine is increasing in recent years. Moreover, as microbial infections present a crucial complication in various treatments, their antimicrobial properties are gaining more attention. The antimicrobial peptides (AMPs) are secreted by cells from various perinatal derivatives, including human amnio-chorionic membrane (hACM), human amniotic membrane (hAM), and human chorionic membrane (hCM). By exhibiting antibacterial, antifungal, antiviral, and antiprotozoal activities and immunomodulatory activities, they contribute to ensuring a healthy pregnancy and preventing complications. Several research groups investigated the antimicrobial properties of hACM, hAM, and hCM and their derivatives. These studies advanced basic knowledge of antimicrobial properties of perinatal derivatives and also provided an important insight into the potential of utilizing their antimicrobial properties in a clinical setting. After surveying the studies presenting assays on antimicrobial activity of hACM, hAM, and hCM, we identified several considerations to be taken into account when planning future studies and eventual translation of fetal membranes and their derivatives as antimicrobial agents from bench to bedside. Namely, (1) the standardization of hACM, hAM, and hCM preparation to guarantee rigorous antimicrobial activity, (2) standardization of the antimicrobial susceptibility testing methods to enable comparison of results between various studies, (3) investigation of the antimicrobial properties of fetal membranes and their derivatives in the in vivo setting, and (4) designation of donor criteria that enable the optimal donor selection. By taking these considerations into account, future studies will provide crucial information that will enable reaching the optimal treatment outcomes using the fetal membranes and their derivatives as antimicrobial agents.

Amnijska membrana (AM) predstavlja alternativno možnost za zdravljenje bakterijskih okužb, med njimi tudi okužb urinarnega trakta. Z difuzijskim testom na agarju smo testirali učinek homogenata humane amnijske membrane (hAM), pripravljenega na različne načine, na rast sevov bakterije Escherichia coli DH5α, DL88, DL90, DL94, DL101, DL102 ter na rast bakterij Staphylococcus aureus (S. aureus), Enterobacter species (Enterobacter sp.) in Serratia mercescens (S. mercescens). Ugotovili smo, da izbira homogenizatorja ter sam postopek priprave hAM (npr. filtriranje skozi 0,22 µm filter), vpliva na protimikrobni učinek homogenata hAM. S poliakrilamidno gelsko elektroforezo v prisotnosti natrijevega dodecilsulfata (SDS-PAGE) smo ugotovili, da obstajajo razlike v proteinski sestavi homogenatov hAM, pripravljenih na različne načine. Pridobljeni rezultati o aktivnosti pripravkov iz hAM so nam tako razkrili, katerim korakom pri pripravi homogenata hAM je treba nameniti posebno pozornost v nadaljnjih raziskavah. Amniotic membrane (AM) is an alternative solution for the treatment of bacterial infections, including urinary tract infections. Antimicrobial effect of various preparations of human amniotic membrane (hAM) on different bacterial strains was tested using the diffusion method. Included were Escherichia coli strains DH5α, DL88, DL90, DL94, DL101, DL102 and bacteria Staphylococcus aureus (S. aureus), Enterobacter species (Enterobacter sp.) and Serratia mercescens (S. mercescens). We found that the choice of homogenizer and the process of hAM preparation (e.g. filtration through a 0.22 μm filter) influenced the antimicrobial effect of hAM homogenate. With sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) we found differences in the composition of the different hAM preparations. The obtained results on the activity of hAM preparations thus revealed to which steps in the preparation of the hAM homogenate special attention in further research should be given.

Prokaz primjene amnijske membrane u bolestima površine oka.

Urinary tract infections are among the most common bacterial infections in humans. Moreover, they are highly recurrent and increasingly often resistant to antibiotics. The antimicrobial properties of the amniotic membrane (AM), the innermost layer of fetal membranes, have been briefly reported in the literature, however, the results of published studies are often inconsistent and unclear; moreover, its effect on uropathogenic bacteria has not yet been investigated. Further, there is no data in the literature about the effect of AM preparation and storage on its antimicrobial properties. To examine the impact of several preparation procedures on the antimicrobial properties of AM, we prepared patches and homogenates of fresh (fAM) and cryopreserved (cAM) human AM and tested them on 14 selected Gram-positive and Gram-negative uropathogenic bacteria. By employing novel antimicrobial efficiency assays we showed that fAM and cAM homogenates have broad-spectrum antimicrobial activity against all here tested uropathogenic bacteria, except for Serratia marcescens. Moreover, they had a potent effect also on the multiple-resistant clinical strains of uropathogenic Escherichia coli. Interestingly, the patches of fAM and cAM had no antimicrobial effect on any of the tested strains. We therefore prepared and stored AM patches according to the standard procedure for clinical use in ophthalmology, which includes the cryopreservation of antibiotic-treated AM, and performed antimicrobial efficiency assays. Our findings suggest that the ultrastructure of AM patches could enable the retention of added antibiotics. In addition, we also prepared gentamicin-resistant uropathogenic E. coli strains, which confirmed that the antimicrobial effect of antibiotic-treated AM patches can be attributed to the antibiotic alone. To summarize, here we describe novel protocols for preparation and storage of AM to ensure the preservation of its antimicrobial factors. Moreover, we describe the mechanism of AM retention of antibiotics, based on which the AM could potentially be used as a drug delivery vehicle in future clinically applicable approaches.

Bolezni sečil zaradi staranja prebivalstva predstavljajo vedno večji izziv. Z namenom iskanja novih terapevtskih pristopov, smo v doktorski disertaciji analizirali učinek pripravkov iz humane amnijske membrane (AM) na rakave urotelijske celice ter uropatogene bakterije. Z različnimi celično-biološkimi, molekularnimi in mikrobiološkimi pristopi smo analizirali učinek nosilcev iz AM, homogenata AM in celic AM na celice T24, ki izvirajo iz humane mišično invazivne urotelijske neoplazije. Ugotovili smo, da 1) nosilci iz AM zavirajo proliferacijo in zmanjšajo invazivni potencial celic T24 ter prispevajo k njihovi mezenhimsko-epitelijski transformaciji, 2) homogenat AM zavira proliferacijo in povzroča luščenje celic T24 ter preprečuje njihovo pritrjanje na podlago in 3) da celice AM zavirajo proliferacijo celic T24. Protimikrobni učinek pripravkov iz AM smo testirali na 25-ih sevih uropatogenih bakterijskih vrst. Dokazali smo, da koščki sveže (svAM) in krioshranjene AM (kAM) nimajo protimikrobnega učinka, medtem ko homogenati svAM in kAM povzročijo cono protimikrobnega učinka pri 16 od 25 testiranih sevov uropatogenih bakterijskih vrst. Protimikrobno delovanje homogenata kAM smo dokazali tudi na z bakterijami okuženih modelih normalnega in rakavo spremenjenega urotelija in vitro. Način priprave ter shranjevanja AM vpliva na obseg njenega protimikrobnega učinka. Ugotovitve, da pripravki iz AM delujejo zaviralno na rakave urotelijske celice in izbrane uropatogene seve uropatogenih bakterijskih vrst, pomembno prispevajo k razumevanju delovanja AM ter predstavljajo izhodišče za nadaljnje študije, ki bodo vrednotile možnosti uporabe AM v urologiji. The urinary tract diseases pose an increasing challenge due to the aging of population. We aimed to find new therapeutic approaches, thus we analysed the effect of human amniotic membrane-derived (AM) preparations on cancer urothelial cells and uropathogenic bacteria. Using different cell-biological, molecular and microbiological approaches we analysed the effect of AM scaffolds, AM homogenate and AM-derived cells on T24 cells, derived from human muscle-invasive bladder cancer. We demonstrated that 1) AM scaffolds diminish the proliferation and reduce the invasive potential of T24 cells and contribute to their mesenchymal-to-epithelial transformation, 2) AM homogenate diminishes the proliferation and causes detachment of T24 cells and prevents their attachment to the surface and 3) AM-derived cells diminish the proliferation of T24 cells. The antimicrobial effect of AM preparations was tested on 25 strains of uropathogenic bacterial species. We demonstrated that patches of fresh (fAM) and cryopreserved AM (cAM) have no antimicrobial effect, while the fAM and cAM homogenates have antimicrobial effect on 16 out of 25 tested strains of uropathogenic bacterial species. Moreover, cAM homogenate has antimicrobial effect also on bacteria-infected in vitro models of normal and cancerous urothelium. The manner of preparation and storage of AM affects the range of its antimicrobial effect. Findings that AM preparations inhibit cancer urothelial cells and selected strains of uropathogenic bacteria, importantly contribute to to the knowledge of AM activity and serve as a baseline for studies that will evaluate the use of AM in urology.

Limbalne epitelijske matične celice (LEMC) se nahajajo v roženičnem limbusu in omogočajo obnovo roženičnega epitelija. Okvara in pomanjkanje teh celic vodi v boleznsko stanje. Eden od načinov zdravljenja pomanjkanja LEMC je s presaditvijo vzgojenega limbalnega tkiva v kulturi. Določali smo imunogenost gojenega roženičnega tkiva iz keratolimbalnih eksplantov kadavrskih roženic do tretje pasaže na plastiki in intaktni amnijski membrani (AM). S pretočno citometrijo smo določali prisotnost dveh podtipov dendritičnih celic (DC), ki smo jih ločili po izražanju naslednjih označevalcev, za plazmacitoidne DC (pDC): CD123+, CD303+, CD11c-, in za mielodine DC (mDC): CD11c+, CD123-. Merili smo tudi izražanje molekule MHC II, kostimulatornih molekul CD80 in CD86 ter aktivacijske molekule CD83. Merili smo še izražanje molekul CK12 in ABCB5. Prve so značilne za populacijo zrelih epitelijskih roženičnih celic, druge pa za pa populacijo LEMC. Za vse parametre smo naredili modele napovedi povprečnih deležev po pasažah. Pri vseh parametrih se je izkazalo, da razlika med rastjo celic na plastiki in na AM za gojenje ni statistično značilna (p>0,05). Z višanjem pasaž smo opazili trend znižanja deleža pDC in mDC, vendar pa vrednosti niso bile statistično značilne. Trdimo lahko, da je padec števila zrelih epitelijskih roženičnih celic iz prve v drugo (p=0,007) in iz druge v tretjo pasažo (p=0,01) statistično značilen. Prav tako je statistično značilen padec LEMC iz prve v drugo pasažo (p=0,02). Glede na rezultate menimo, da je na obeh podlagah smiselno gojiti celice do vključno prve pasaže, tako dobimo tkivo, ki ima zadostno količino epitelijskih roženičnih celic, LEMC in zmanjšan delež mDC in pDC. Limbal epithelial stem cells (LESCs) are found in the corneal limbus and are important for corneal epithelium renewal. Lack of these cells leads to a limbal stem cell deficiency (LSCD). We determined the immunogenicity of cultivated tissue from keratolimbal explants of cadaveric corneas up to the third passage grown on plastics and on intact amniotic membrane (AM). Flow cytometry was used to determine the presence of two subtypes of dendritic cells (DC), which were determined by measuring the expression of the following markers: for plasmacitoid DC (pDC): CD123+, CD303+, CD11c-, and for myeloid DC (mDC): CD11c+, CD123-. We also measured the expression of MHC II molecule, the CD80 and CD86 costumulatory molecules, and CD83 activation molecule. Populaton of mature epithelial cells was determined by measuring the expression of CK12, and the population of LESC by the expression of ABCB5. Models of forecasts of average percentage by passages were made. The difference between plastics and AM was not statistically significant (p>0.05) for any measured parameter. Increasing passages reduced the percentage of pDC and mDC, but the values were not statistically significant. The average percentage of mature epithelial corneal cells fell from first to second (p=0.007) and from second to third passage (p=0.01). The average percentage of LEMC from the first to the second passage (p=0.02) also dropped. According to our results, it is best to cultivate the cells up to and including the first passage for both substrates, thus obtaining a tissue that has a sufficient amount of epithelial corneal cells, LEMC, and a reduced proportion of mDC and pDC.

U okviru veterinarske medicinine biomedicinski potencijal amnijske membrane vrlo se rijetko koristi. Stoga je u ovom radu ispitivana vijabilnost limbalnih stanica psa in vitro kultiviranih na amnijskoj membrani porijeklom od zdrave kuje dobivene pri carskom rezu. Mjereno fluorimetrijskim Cell Titer Blue testom, pokazano je da nema statistički značajnih razlika između vijabilnosti stanica uzgojenih na plastici (intenzitet fluorescencije 5725-6000) i onih uzgojenih na amnijskoj membrani (intenzitet fluorescencije 5815-5950). Rezulati prikazanih istraživanja, iako dobiveni na statistički malom broju uzoraka, u okviru veterinarske medicine otvaraju nove mogućnosti primjene tkivnog inženjerstva u veterinarskoj oftamologiji.

The aim of the study was to determine whether amniotic membrane could be used to treat neuroparalytic keratopathy. Amniotic membrane transplantation was performed in 19 patients with neuroparalytic ulcer. All patients were younger than 61 and all developed corneal problems due to interruption of the sensory afferent fiber innervating the cornea. In all patients, the follow up period was longer than 18 months. Postoperatively, all patients underwent fluorescein test and biomicroscope examination as well as subjective pain evaluation. Postoperatively, the pain was reduced in study patients. In one patient, retransplantation for disease recurrence was performed two months after the first operation. After the second operation, the pain was minimized for more than 18 months. Amniotic membrane transplantation appears to be a good method for pain reduction and better epithelial healing in patients with neuroparalytic keratopathy., Cilj rada bio je utvrditi može li se amnijska membrana rabiti u liječenju neuroparalitične keratopatije. Transplantacija amnijske membrane je učinjena kod 19 bolesnika s neuroparalitičnim ulkusom. Svi bolesnici bili su mlađi od 61 godine i kod svih su se problemi s rožnicom razvili kao rezultat prekida senzornih aferentnih vlakana koja inerviraju rožnicu. Razdoblje praćenja je kod svih bolesnika bio duži od 18 mjeseci. Poslijeoperacijski je kod svih bolesnika učinjen fluoresceinski test i biomikroskopski pregled, kao i subjektivna procjena boli. Nakon operacije bol je bila smanjena kod svih bolesnika. Kod jednog bolesnika učinjena je druga transplantacija, dva mjeseca nakon prve, zbog recidiva. Nakon druge operacije bol je bila smanjena više od 18 mjeseci. Transplantacija amnijske membrane čini se kao dobra metoda smanjenja boli i postizanja boljeg epitelijalnog cijeljenja kod bolesnika s neuroparalitičnom keratopatijom.

Svrha. Amnijska membrana se pokazala učinkovita u liječenju defekata epitela rožnice, bulozne keratopatije i ostalih bolesti prednje površine oka. Većina se citokina (BFF, TGF-b) koji se luče iz amnija nalazi u epitelu. Do sada uobičajen način primjene amnija bio je njegovo prišivanje epitelnom stranom &#8220 ; ; prema gore&#8221 ; ; . U ovoj se studiji uspoređuju prvi rezultati transplantacije amnija metodom &#8220 ; ; prema dolje&#8221 ; ; s rezultatima klasičnog pristupa. Metode. Pacijenti s perzistentnom erozijom epitela (n=10) i buloznom keratopatijom (n=16) nasumce su podijeljeni u dvije grupe od kojih je grupi 1) transplantirana amnijska membrana s epitelnom stranom usmjerenom &#8220 ; ; prema gore&#8221 ; ; a grupi 2) epitelnom stranom usmjerenom ka rožnici ili &#8220 ; ; prema dolje&#8221 ; ; . Amnijske membrane priređene su u Lions hrvatskoj očnoj banci i čuvane na -20°C. Rezultati. Cijeljenje epitelnih defekata postiglo se u 80% slučajeva u obje grupe. Kod bulozne pseudofakije bol je smanjena u 90% slučajeva bez obzira na orijentaciju membrane. Zaključci. Transplantacija amnijske membrane epitelom &#8220 ; ; prema dolje&#8221 ; ; kako bi se prevenirala bol (u bulozne keratopatije) ili postiglo zacijeljenje erozija rožnice čini se jednako učinkovita kao metoda epitelnom stranom &#8220 ; ; prema gore&#8221 ; ; .

Purpose: Amniotic membrane shows efficiency in treatment of corneal epithelial defects, bullos keratopathy and other ocular surface diseases. Most of the cytokines (BFF, TGF-β ) that are secreted from the amniotic membrane are located in the epythelium. Till now, common way for the amniotic membrane transplantation was with the epythelial side "UP". In this study we compared first results of the amniotic membrane transplantation with the epythelial side "DOWN" with results from the clasical aproach. Methods: Patients with the permanent epythelial erosions (n=14), corneal ulcer (n=6) and bullos keratopathy (n=16) were randomly devided in two groups. In first group of patients amniotic membrane was transplanted with the epythelial side "UP", in second group amniotic membrane was transplanted with the epythelial side "DOWN". Amniotic membranes were prepared in Lions Croatian Eye Bank and stored at -20 oC. Results: Reepithtelisation was succesfull in 80% of cases in both groups with epythelial erosions, while success rate of treatment in patients with corneal ulcer was 56% in both groups. In patients with bullos keratopathy reduction of pain was achived in 90%, and pain reduction don't depend on the transplant orientation. Conclusion: Amniotic membrane transplantation with epithelial side "UP" is equaly effective as transplantation with epithelial side "DOWN" in treatment of corneal epithelial defects and corneal ulcers, as well as in pain reduction in patients with bullos keratopathy.

CILJ: Procjena sigurnosti i uspješnosti transplantacije amnijske membrane u svrhu rekonstrukcije prednjeg segmenta oka. METODE: Amnijska membrana dobivena je od placente neposredno nakon izvršenog carskog reza. Preparacija i pohrana vršena je u Lions Hrvatskoj očnoj banci prema međunarodnom protokolu očnih banaka. U razdoblju od 1998. do 1999. godine učinjena je transplantacija amnijske membrane na 42 oka. Indikacije za operaciju bile su: skupina A: perzistentni ulkus rožnice (rezistentan na svu dosadašnju terapiju, n=12), grupa B: simptomatska bulozna keratopatija (n=10), grupa C: opekline oka sa stvaranjem simblefarona (transplantaciji amnijske membrane prethodila je transplantacija rožnice, n=3), grupa D: rekurentni pterigij (n=5), grupa E: erozija rožnice (n=12). REZULTATI: A) Cijeljenje ulkusa rožnice zabilježeno je u 50% očiju, a značajno smanjenje upale u 100%, B) 100% očiju postalo je asimptomatsko, C) transplantat rožnice ostao je proziran, epiteliziran, a pokretljivost oka zadovoljavajuća, D) prevencija ponovnog nastanka pterigija u 70% očiju, E) reepitelizacija u 70% očiju ZAKLJUČAK: Transplantacija amnijske membrane pokazala se djelotvornom metodom u liječenju određenih bolesti rožnice. Na očima kod kojih je učinjena transplantacija amnijske membrane došlo je do smanjenja upale i vaskularizacije, što rekonstrukciju prednjeg očnog segmenta čini uspješnijom.

Transplantacija amnijske membrane koristi se u terapiji raznih bolesti ovršine oka