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Since only few reported studies propose anti-vascular endothelial growth factor (anti-VEGF) delivery through electrospun scaffolds, this study greatly contributes to the potential prevention of patient’s vision loss, as it explores electrospun polycaprolactone (PCL) coated with anti-VEGF for the blockage of abnormal cornea vascularization. In terms of physicochemical properties, the biological component increased the PCL scaffold fiber diameter (by ~24%) and pore area (by ~82%), while ut slightly reduced its total porosity as the anti-VEGF solution filled the voids of the microfibrous structure. The addition of the anti-VEGF increased the scaffold stiffness almost three-fold at both strains of 5 and 10%, as well as its biodegradation rate (~36% after 60 days) with a sustained release profile after Day 4 of phosphate buffered saline incubation. In terms of scaffold application function, the PCL/Anti-VEGF scaffold proved to be more favorable for the adhesion of cultured limbal stem cells (LSCs) ; this was confirmed by the SEM images, where the cells showed flat and elongated conformations. Further support of the LSC growth and proliferation was con-firmed by the identified p63 and CK3 markers after cell staining. These results demonstrate the advantageous effect of the surface-adsorbed anti-VEGF to stop vision loss and help damaged corneal tissue repair.

The aim of this study is to present development of tissue engineering and clinical application of cultured epidermal autografts (CEA). Advanced therapy medicinal products (ATMPs) are medicines for humans that include gene, somatic cell and tissue-engineered therapeutic products. Cultured keratinocytes regenerate epithelium and belong to the category of ATMP as tissue-engineered products. The development of ATMPs in Croatia began during 2002 in collaboration with the Ruđer Bošković Institute, the Clinic of Traumatology and the Children's Hospital Zagreb on the project Production of skin grafts in vitro. Engineering Laboratory was built in May 2005 in accordance with Good Manufacturing Practice and clean room technology. Tissue and Cell Bank (TCB) was established during 2007. The procedure includes isolation of keratinocytes from the skin biopsy (about 4-6 cm2/0.3 mm thick) after which they are seeded onto a feeder layer of 3T3 cells and incubated at 37 °C, 5 % CO2. Preparation of the optimal number of grafts is accomplished within 3- 4 weeks depending on the area of the injury. Immunocytochemistry (ICC) combines histological, immunological and biochemical techniques in the identification of specific tissue components. Using flow cytometry enabled high-throughput analysis of keratinocytes and residual feeder cells. Detection of bacterial endotoxins was determined using a Lysate of Amebocyte Limulus test. According to the EMA guidance the recommended endotoxin limit for release testing is ≤ 0.5 EU/mL (endotoxin units). Cell growth was monitored on a daily basis on microscope and analyzed on 14th day for evaluation of colony- forming efficiency (CFE). CFE was calculated as the number of colonies divided by the total number of seeded cells (1000 or 1500) and multiplied by 100 to express the result as a percentage. Quality control involves potency (yield, viability, CFE), purity (ΔNp63, CK AE1/AE3, CK5/6, CK14, CK19, vimentin), impurity (remaining 3T3 cells) and safety (sterility, mycoplasma, bacterial endotoxins).The first successful production of epidermal grafts began in the Clinic of Traumatology in September 2002 with 10 epidermal grafts of 700 cm2. This retrospective analysis spans a period from February 2002 to October 2003. The project included donors (n=15), from 2 to 66 years old with total 92 CEA. Microbiological control has proven the sterility of all keratinocytes, cell media as well as epidermal transplants. From July 2007 to March 2022 in TCB, donors (n=62) were from 1 to 74 years old with total 2235 CEA from which 89.2 % were transplanted and 10.8 % discarded. The most common reasons for discardment were patient’s death, initial microbiological contamination (Acinetobacter baumannii, Cutibacterium acnes, Micrococcus spp., Pseudomonas aeruginosa, Staphylococcus capitis, Staphylococus epidermidis and other coagulase negative staphylococcus, etc.) and technical reasons (problem with feeder layer and decontamination of skin biopsy). Growth media from CEA was monitored for bacterial endotoxins prior to clinical application. Our results were < 0.125 EU/mL. CFE for 1000 cells was ≈ 9, 3 % and for 1500 cells was 8.3-15.1 %. ICC analysis showed 100 % epithelial cell marker CKAE1/AE3 positive cells (A), 100 % CK14 expressed in mitotically active basal layer cells (B), without CK19 positive differentiated keratinocytes (C), with 22.5 % proliferation marker Ki-67 positive cells (D) and 32 % vimentin positive cells represent pool of good quality of keratinocytes. Flow cytometry analysis shows high percentage of ΔNp63 (99.6 %) positive cells and low percentage of anti-feeder cells (1.66 %) positive cells ensure a good quality of CEA for the transplantation. Keratinocytes prepared as epidermal grafts or suspension with fibrin glue contributed to the survival of severely burned patients.

Spongy bone is used in reconstructive procedures as a valuable material for bone regeneration and replacement. The aim was to examine the aseptic procurement and cutting of femoral head fragments in the operating room, the sterility of spongy bone obtained in the microbiological safety cabinet (MSC) and to show histological structure and vitality of tissue. During the implantation of a total hip endoprosthesis in living donors, the femoral heads were removed and cut into fragments. A swab for microbiological and a biopsy for pathohistological analysis were sampled from each head. Fragments were processed in MSC and the sterility of spongiosis was controlled by analysing duplicate swabs and biopsies by direct inoculation in a liquid medium for anaerobic and aerobic bacteria and fungi. Sterility of environmental conditions (surface, air and operator’s fingers) was controlled by contact and sediment plates. 27 samples were microbiologically analysed: 16 from the operating room (biological n=10, environmental n=6) and 11 from MSC (biological n=7, environmental n=4). A 100 % sterile result of bone tissue and operator's fingers was obtained in the operating room and the results of air control were within acceptable limits. All results sampled in the MSC were 100 % sterile. Pathohistological analysis confirmed vital osteocytes in the lacunae of the circumferential lamellae, individual osteoclasts and numerous osteoblasts in the surface of the bone without any pathological changes.

UVOD: Unazad 15 godina jedna od osnovnih djelatnosti Banke tkiva i stanica je prikupljanje koštanih presadaka alogenih darivatelja (glave bedrene kosti). Koštani presaci se uzimaju tijekom operativnog zahvata ugradnje TEP-a kuka. Tijekom zahvata operater procjenjuje kvalitetu koštanog presatka i donosi konačnu odluku o pohrani tkiva. Prilikom uzimanja, u operacijskoj sali, svim koštanim presacima uzimaju se uzorci za mikrobiološku kontrolu. Presaci se pohranjuju i čuvaju u zamrzivačima banke na temperaturi -80 °C s rokom trajanja 5 godina. Od 2012. godine u suradnji s Klinikom za ženske bolesti i porodništvo KBC Sestre milosrdnice, razvijena je i djelatnost prikupljanja posteljica i pripreme amnijskih presadaka. Posteljice se uzimaju isključivo tijekom poroda carskim rezom od alogenih darivateljica. Nakon uzimanja, daljnja obrada posteljice provodi se u čistim prostorima (prostori s najvišim stupnjem mikrobiološke čistoće) koji se nalaze u Klinici za traumatologjiu. Od plodnih ovoja se odvaja amnijska membrana koja prolazi proces dekontaminacije te se reže na presatke različitih dimenzija. Presaci amnijske membrane čuvaju se u krioprezervansu u zamrzivačima na temperaturi -80 °C s rokom trajanja 2 godine. Svi darivatelji prije darivanja obavezno prolaze kroz strogi proces odabira i eliminacije te se nakon darivanja testiraju na krvlju prenosive bolesti kako je propisano nacionalnim i europskim zakonskim propisima. REZULTATI: Koštani presaci se koriste u rekonstruktivnim zahvatima ortopedske kirurgije u slučajevima kada se autotransplantatom ili umjetnim materijalom ne mogu postići željeni rezultati. Amnijski presaci se najčešće koriste u oftalmologiji za liječenje ozljeda rožnice. Unazad nekoliko godina počeli su se koristiti i za liječenje ozljeda kože (npr. opekline, avulzije kože i rane koje teško cijele). Svi primatelji presadaka se prate nakon primjene radi uvida u ishod liječenja i prihvaćanja transplantata. ZAKLJUČAK: Tijekom dugogodišnjeg praćenja rezultata liječenja navedenim presacima nije zabilježena niti jedna neželjena reakcija kod primatelja presadaka. Zbog navedenog zaključujemo da su naši presaci kvalitetni i sigurni za kliničku primjenu.

UVOD: Banka tkiva i stanica djeluje u Klinici za traumatologiju od 2005., a od rujna 2019. godine je dio Zavoda za transfuzijsku i regenerativnu medicinu KBC Sestre milosrdnice. Osnovne djelatnosti banke su tkivno bankarstvo (prikupljanje, uzimanje, očuvanje, pohrana i raspodjela glava bedrene kosti uzetih od živih alogenih darivatelja i amnijskih presadaka pripremljenih od posteljica živih alogenih darivateljica) i tkivno inženjerstvo (proizvodnja lijekova za naprednu terapiju, engl. Advanced Therapy Medicinal Products – ATMP), tj. uzgoj keratinocita i limbalnih stanica in vitro za autolognu primjenu. REZULTATI: Glava bedrene kosti uzima se od živih darivatelja tijekom operacije ugradnje TEP-a kuka.. Provodi se detaljan pregled darivatelja, mikrobiološka analiza brisa glave koji se uzima tijekom operacijskog zahvata prije pohrane u sterilnu posudu te analiza krvi darivatelja na krvlju prenosive bolesti. U tablici 1. je plavom bojom prikazan broj prikupljenih presadaka, crvenom broj presadaka za kliničku primjenu, a zelenom broj primijenjenih presadaka godišnje od 2007. do 2021. Do 2020. najčešći razlog uništenja presadaka je bio pozitivan mikrobiološki nalaz brisa, a unazad dvije godine i neprikladnost darivatelja uočena nakon darivanja. Amnijski presaci se proizvode od posteljica živih darivateljica uzetih tijekom carskog reza.. Također se provodi detaljan pregled darivateljica, mikrobiološke i histološke analize tkiva te analiza krvi. U tablici 2. je plavom bojom prikazan broj proizvedenih presadaka, crvenom broj presadak za kliničku primjenu, a zelenom broj primijenjenih presadaka godišnje od 2012. do 2021. Spongioza glave bedrene kosti se koristi kod koštanih defekata dok se amnijski presaci koriste kod teških ozljeda rožnice i kože. Uzgoj autolognih keratinocita i limbalnih stanica in vitro provodi se u čistim sobama. Keratinociti se primijenjuju kodopeklina drugog i trećeg stupnja velike tjelesne površine, a limbalni presaci kod potpunog deficita limbalnih stanica. ZAKLJUČAK: Regenerativna medicina predstavlja budućnost medicine u obnovi strukture i funkcije teško oštećenih tkiva i organa.

Na kongresu su prikazani naši rezultati kod transplantacije uzgojenih autolognih limbalnih stanica u liječenju kombustije oka

The Tissue and Cell Bank is a hospital unit of University Hospital Center “Sestre milosrdnice” in Zagreb that has laboratories with cleanrooms. The Tissue and cell bank, in daily routine work, is divided into tissue engineering and tissue banking. It also has the human resources required for donor evaluation, testing, processing, preservation, storage and distribution of human tissues for transplantation. Our Department of transfusion and regenerative medicine has established a unit for Quality Management that deals with aspects such as quality control in all segments of work in tissue banking and cell therapy. Cultivation of limbal stem cells is a well established method of cellular therapy for patients with limbal epithelial stem cell deficiency caused by chemical burns, thermal injuries or chronic immune inflammatory diseases. My presentation is going to focus on the analysis of human limbal cells and their characteristics evaluated for transplantation, followed by different kinds of methods like colony forming efficiency, flow cytometry, histopathology, immunocytochemistry, immunohistochemistry, florescence immunocytochemistry, ect. Growth media from limbal cells culture, were monitored for bacterial endotoxin before to clinical application.

Aim was to investigate the impact of synthetic electrospun polyurethane and polycaprolactone nanoscaffolds, before and after hydrolytic surface modification, on viability and differentiation of cultured human eye epithelial cells, in comparison with natural scaffolds: fibrin and human amniotic membrane. Human placenta was taken at elective caesarean delivery. Fibrin scaffolds were prepared from commercial fibrin glue kits. Nanoscaffolds were fabricated by electrospinning. Limbal cells were isolated from surpluses of human cadaveric cornea and seeded on feeder 3T3 cells. The scaffolds used for viability testing and immunofluorescence analysis were: amniotic membrane, fibrin, polyurethane (PU) and polycaprolactone (PCL) nanoscaffolds, with or without prior NaOH treatment. SEM photographs of all tested scaffolds showed good colony spreading of seeded limbal cells. In regard to viability performance there was statistically significant difference between cells with highest viability cultured on tissue culture plastic and cells cultured on all other scaffolds. On the other hand, electrospun PU, PCL and electrospun PCL treated with NaOH had more than 80% of limbal cells positive for stem cell marker p63 compared to only 27%of p63 positive cells on fibrin. Natural scaffolds, fibrin and amniotic membrane, showed better cell viability compared to electrospun scaffolds. On contrary, high percentages of p63 positive cells on these scaffolds still makes them good candidates as efficient delivery systems for therapeutic purposes.