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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.

In July 2014 production facility in our Tissue Bank at Department of Traumatology, University Hospital Centre Sestre milosrdnice, was officially licensed by Croatian Competent Authority (CA) for the production of cultured autologous keratinocytes for treatment of burns under the hospital exemption rule. Early coverage of the wound bed in burned patients is mandatory for the treatment. Glycerol preserved allografts (GPAs) are often used for this purpose, especially in extensive burns. Since 2016 we are importing GPAs from Banc de Sang i Teixits, Barcelona. Our clinical experience is that GPAs create a good environment for subsequent application of cultured autologous keratinocytes. In 2017 we decided to make a small pilot study in our University Hospital Centre involving explantation and processing of cadaveric skin. After the Hospital Ethics Committee approval and permission from our CA we have explanted cadaver skin from one Donor after Brain Death (DBD) and the two Donors after Cardiac Death (DCD). DBD and DCD donors were from 35 to 51 years old. Average cooling time of the bodies before the commencement of explantation was less than 24 hours. Thorough evaluation of a donors' social and medical history as well as blood tests were performed. Explantations were carried out in our hospital in operating theater and in autopsy room, depending on the type of the donor. Explantation team consisted of one plastic surgeon and three medical technicians. Immediately after the excision, the skin was put in solution of 50% of sterile glycerol with antibiotics and transported to the tissue bank for further processing. The largest skin quantity obtained from one donor was 1000 cm2. Skin grafts were processed in clean room, in class A with class B background and finally preserved in solution of 85% of glycerol at +4°C. Skin bioptates were taken for additional microbiological control at regular intervals up to one year after the explantation. GPAs processed in this pilot project served only for the educational and validation purposes. Additional efforts must be made in order to further educate and practice explantation team in aseptic techniques and in obtaining larger area of the skin with more uniform size.

Functionalized electrospun materials have many applications one of which includes biomedicine or tissue engineering. These materials act as scaffolds as can simulate the extracellular matrix in tissues thus giving support to cells growth and finally helping tissue regeneration. This study gives an insight into the research concerning development of this type of materials as part of the Croatian Science Foundation project. Electrospun composite scaffolds were prepared by electrospinning of neat polycaprolactone (PCL), as well as PCL blended with an antibiotic or silk fibroin separately and finally by electrospinning both PCL/fibroin and PCL/antibiotic within a layered configuration. The scaffolds were analyzed in regard to their morphology, total porosity, surface wettability and tensile behaviour taking into account the type of filler and layered structure. Firstly the addition of the antibiotic as well as silk fibroin both resulted in the reduction of the water contact angle on the surface of the hydrophobic PCL. Thus, a hydrophilic surface is of paramount importance as it improves scaffolds cells adhesion. The addition of the fillers, antibiotic and silk fibroin, resulted in PCL fibers diameter reduction by 28% and increase by 224%, respectively. Fibers deformations or beads were present in case of the neat PCL and the PCL/fibroin fibers. The biggest pores area were in case of the PCL/fibroin of 8.13±6.45 µm2. The total porosity reduced gradually with the addition of the fillers, resulting in the lowest value in case of the layered scaffolds (from 94.2 to 68.5%). The tensile strength of the electrospun scaffolds improved with the addition of the antibiotic and in case of the layered structure, resulting in the maximum value of 260.33±60.53 cN/mm2. These results suggest that the nature of the filler affects its miscibility with the polymer and electrospinning jet stretching and both porosity and tensile strength are affected by the scaffolds composition and layered structure. In this way by variating the composition and physical structure, scaffolds can be designed according to requirements in tissue therapy and repair.

For centuries silk fibroin has been used as a textile fiber. Beyond textiles, silk fibroin has also been used in biomedical applications as a surgical material for decades. Because of his good mechanical properties, biocompatibility and biodegradability silk fibroin is being further developed for various emerging applications. In this work, tissue engineering scaffolds composed of polycaprolactone (PCL) and silk fibroin (SF) were prepared by electrospinning technique. Morphology, chemical composition, thermal properties, thermal stability and hydrophobicity of the PCL/SF scaffolds were characterized to evaluate the effect of SF by means of scanning electron microscopy (SEM), Fourier transform infrared spectra (FTIR), differential scanning calorimetry (DSC), thermogravimetric analysis (TGA) and water contact angle analysis. Electrospinning resulted in uniform fibers morphology, with thinner fibers in case of the PCL/SF scaffolds and a homogeneous SF distribution throughout the fibers. The FTIR results showed that the silk fibroin was successfully introduced to the PCL scaffolds. The hydrophilicity of the PCL modified scaffolds was enhanced. Results obtained with DSC analysis showed that with addition of the silk fibroin, glass transition temperature of the PCL increases, while the melting temperature and degree of crystallinity decreases, because of the less organized crystal structure with addition of silk fibroin. The thermal stability of the PCL/SF scaffolds also decreased with the addition of the SF.

The study focuses on the design and evaluation of electrospun dual function scaffolds with the ability to heal inflammation in skin and eye tissues and to provide topographical cues for cells penetration into the scaffolds depth. For this purpose scaffolds were loaded with antibiotic and electrospun on 3D printed collectors with purpose made geometry.

Authors’ very first experience with application of glycerol preserved allografts (GPAs) in acute burn treatment applied in a 7-year-old boy with 93% TBSA IIIrd degree flame burn. After excision of the necrotic tissue during the initial 4 days, INTEGRA DRT was applied on the extremities. Given the fact that the take rate of INTEGRA DRT was around 30%, allogenic skin grafts were imported from the Banc de Sang i Teixits - Barcelona and grafted onto the wound bed, 14 days after the patient’s admission. Donor sites on the scalp and the foot were covered with amniotic membranes (AM) from the local Tissue Bank. During 4 months of hospitalization, GPAs were applied 6 times (7, 000 cm2). AM promoted faster healing in small areas of IInd degree burns. Cadaveric skin grafts enabled the preservation of the wound bed for subsequent autologous skin grafting combined with cultured epithelial autografts (CEAs). CEAs were applied in preconfluent phase with two - component fibrin glue (Tisseel, Baxter). The take rate after wound bed conditioning with allogenic skin was around 90%. An episode of mold infection occurred on the area covered with allogenic skin what prompted immediate removal of the grafts and initiation of antifungal therapy. Allogenic skin grafts provided a beneficial temporary wound dressing and enabled a very satisfying take rate of autologous skin grafts. Unfortunately, six months after the admission, when burns were healed, the patient died out of acute heart failure.

This paper work is based on a brief discussion of a recent project proposal, approved by the Croatian Science Foundation. The project proposal focuses on the application of the technique of electrospinning in the development of scaffold prototype to be used for in vitro culture of ocular and skin cells. A brief review was also conducted on recent studies focusing on the usage of electrospinning combined with other advanced techniques for the fabrication of materials used for biomedical applications. The project itself has proposed scaffolds fabrication combining both solution and melt electrospinning.

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The fundamental goal in veterinary ophtamology is application of in vitro grown stem cells/tissues as auto/allo transplants from animal species of interest for veterinary medicine (dog, cat, horse and pig) as model for transplantation research in therapy of corneal defects and deficiences. The aim of this study was to evaluated effectiveness of subconjunctival application of limbal stem cells allo-grafts to dogs in proliferation and circulating immune cell subsets.

Viable Environmental Monitoring (EM) for an ATMP aseptic manufacturing area includes personnel, air and area surfaces control for the presence of microorganisms. Qualification studies and written procedures should exist for the selection of sampling sites, frequencies and modes of microbial testing. For a given class of air cleanness alert and action levels must be predetermined. Microbiological sampling of our personnel, surfaces and the air was carried out using contact and settle plates and active sampling device. Frequencies’ of microbial testing depended on cleanroom category and for A and B class included each working session. Number of sampling locations per room was calculated from square root of cleanroom zone area in m2. Last year we had in A class: 0.6 % of action levels for both, the air and surface testing. B class showed: 5% alerts and 4% action levels for the air samples and 1% of alerts and action levels for surface testing. 8 bacterial and fungal species were isolated from different room areas. The most detected microorganism in A and B (air -39% ; surface -55%), C (air-35% ; surface-48%) and surface of D class (69%) was CoNS. In the air of D class most detected microorganism was Micrococcus spp. (39%). We have determined the microbial distribution in our cleanroom facility. Several bacterial species were identified as the most probable contaminants that could affect our final product – cultivated epithelial autografts (CEA). Knowing the EM data and undertaking appropriate corrective measures the risk of contamination can be significantly reduced.

Femoral head allografts removed as the surgical surplus bone tissue during the total hip arthroplasty procedure represent valuable clinic material, especially if cadaveric grafts are not available. Nevertheless, care must be taken that extensive safety and quality control measures during the femoral heads banking are fulfilled. In the Clinic for traumatology, these allografts are used as the method for repairing large bone defects providing optimal implant fixation and long-term support for the components. We examined the clinical use of femoral head allografts in our Clinic since 2010 in which structural allografts showed best available option for bone defect reconstruction in trauma and orthopaedic, spinal and other surgical procedures. Femoral head allografts were mostly used for different indications of operative grafting procedures in the treatment of proximal and distal tibial and fibular (31, 4%), femoral (29, 27%), humeral (20, 21%), calcaneal, pelvic and vertebral fractures. Proximal tibia fractures included B and C type of fracture were treated with different quality of bone healing results. Femoral head allografts have some drawbacks like lack of the specific osteosynthetic material, low growth potential at recipient’s site and lower quality of the cavernous bone tissue (old donors). Despite all of this, frozen bone allografts provided by our local Tissue Bank are of great importance, particularly in situation where the use of autograft could pose an additional health risk to a patient.

The aim of the study was to investigate the impact of 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. The scaffolds used were: amniotic membrane, fibrin, polyurethane (PU) and polycaprolactone (PCL) nanoscaffolds, with or without prior NaOH treatment. 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. Scaffolds morphological characterization was performed using scanning electron microscopy. Human limbal cells were identified by immunofluorescence confocal laser scanning microscope, using markers for limbal stem cells p63 and differentiated cells of the cornea CK3. There was statistically significant difference in viability of cells cultured on plastic and cells cultured on all other scaffolds. Cells grown on fibrin and amnion showed higher viability compared to the electrospun nanoscaffolds. 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. SEM photographs of all tested scaffolds showed good colony spreading of seeded limbal cells. Although compared to natural carriers, the higher porosity and larger surface of nanoscaffolds and their NaOH treated versions, promised superior cell attachment and spreading, this was not the case. They showed lower cell viability compared to fibrin and plastic. On the contrary, high percentages of p63 positive cells on these scaffolds still makes them good candidates as efficient delivery systems for therapeutic purposes. Modifications in nanoscaffold surface properties should be more thoroughly tested to achieve higher level of viability and proliferation.

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.

U okviru veterinarske medicinine biomedicinski potencijal nanonosača vrlo se rijetko koristi. Stoga je u ovom radu ispitivana vijabilnost limbalnih stanica ovaca in vitro kultiviranih na poliuretanskim nanovlaknima dobivenih metodom elektoispredanja. Mjereno fluorimetrijskim Cell Titer Blue testom, pokazane su statistički značajne razlike između vijabilnosti stanica uzgojenih na plastici (intenzitet fluorescencije 5725-6000) i onih uzgojenih na poliuretanskim nanovlaknima (intenzitet fluorescencije 4000-4500). Premda je statistički gledano, plastika bolji nosač za limbalne stanice od poliuretanskih nanovlakana, rezulati prikazanih istraživanja, iako su dobiveni na statistički malom broju uzoraka, u okviru veterinarske medicine otvaraju nove mogućnosti primjene nanotehnologije u veterinarskoj oftamologiji.

Thermoplastic polyurethane (TPU) has been extensively studied in the field of tissue engineering. Incorporation of titanium dioxide (TiO2) into TPU scaffold benefits further with its antimicrobial, photo-catalytic, anticorrosive and hydrophilic properties that are very desirable in ocular regenerative medicine. We decided to test this new, combined material for biocompatibility with human corneal cells

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 choice of artificial extracellular matrix or scaffold that could offer skin stem cells optimal environment for in vitro growth and differentiation is particularly important. Scaffolds for skin engineering must meet three basic criteria: patient safety, clinical efficacy and convenience of use. The aim of this study was to create in vitro fibrin based substitute with epidermal and dermal component and to evaluate treatment of deep partial and full thickness burns. In this study human skin samples and skin cells from surgical surplus tissue were used. Under highly controlled conditions fibroblasts and keratinocytes were cultured. From commercial fibrin glue kits, fibrin scaffolds were aseptically prepared. For viability tests, scanning electron microscope (SEM) and immunocytochemistry analysis of cells cultured on fibrin scaffold were performed. After hydrosurgical preparation of deep burn necrotic tissue, wound bed was prepared for caltar and skin equivalents. Progress of healing was documented using a drawing chart and photos. In epithelial cultures on feeder layer, keratinocytes had characteristic polygonal morphology, while dermal fibroblasts showed bipolar spindle morphology. SEM images showed good attachment and colony spreading of keratinocytes and fibroblasts on fibrin scaffold. Immunofluorescent staining of keratinocyte cultures on fibrin scaffold showed expressions of CK19 (epithelial stem cells marker), involucrin, and marker of differentiated keratinocytes. Clinical results have clearly shown that appearance of the skin did not differ significantly from areas of transplanted skin using standard techniques. In conclusion, using fibrin-cultured autografts on massive full-thickness burn would result in good healing up to 3 years of follow-up.

Tkivno inženjerstvo je relativno novo multidisciplinarno područje koje povezuje spoznaje i dostignuća tehničkih i prirodnih znanosti, a u medicini ima za cilj u potpunosti obnoviti građu i funkciju tkiva/organa nakon različitih patoloških procesa. Optimizacija in vitro uzgoja staničnog/tkivnog auto/alopresadka limbalnih stanica te njihove terapeutske mogućnosti, poticaj su za osnivanje očne banke prema vrstama životinja od interesa za veterinarsku medicinu te osuvremenjivanje i opremanje postojeće oftalmološke kirurške dvorane i postojećeg staničnog laboratorija u čistu prostoriju klase C ili D (standardi dobre proizvodne prakse, engl. GMP) na Veterinarskom fakultetu Sveučilišta u Zagrebu.

U liječenju teških opeklina od 1981. godine diljem svijeta primjenjuju se višeslojni epitelni presatci uzgojeni po klasičnoj metodi Rheinwalda i Greena. Ova tehnologija razvijena je u RH na Institutu Ruđer Bošković, a od 2007. godine rutinski se izvodi u Banci tkiva Klinike za traumatologiju. No, navedena metoda, uz neospornu vrijednost u spašavanju ljudskih života, ipak ima niz mana. Klasično uzgojenim epitelnim presatcima nedostaje dermalna komponenta, izuzetno su krhki, nepraktični pri manipulaciji i sa varijabilnim postotkom prihvaćanja. Epitel nije u potpunosti nalik epitelu prave kože i od stanica sadrži samo keratinocite. U razvoju novih metoda uzgoja ljudske kože, tkivno inženjerstvo, kao relativno nova grana biomedicine, ima sve veći značaj. Krajnji cilj tkivong inženjerstva je obnova arhitekture i funkcije tkiva/organa nakon različitih patoloških procesa. Kako bi se navedeno postiglo, tkivno inženjerstvo koristi se osnovnim alatom: stanicama, nosačima i faktorima rasta. Glavna funkcija nosača slična je prirodnom ektrsacelularnom matriksu u smislu da potiče proliferaciju, diferencijaciju, i biosintezu stanica. Cilj ovog rada je ispitati i usporediti utjecaj nekoliko tipova nosača koji se koriste u tkivnom inženjerstvu na proliferaciju i diferencijaciju stanica kože kako bi se kreirao puni ekvivalent kože.

The PCL known for its slow biodegradability offers advantageous possibilities for its applications in the field of skin scaffold production. In the current study PCL mats with micro scaled structure were produced via electrospinning and treated with NaOH solution for enhanced cell adhesion. The scaffolds were evaluated as an efficient stem cell growing material by human skin keratinocytes and fibroblasts culture. The cell viability assay (CellTiter-Blue) of the as spun scaffolds was compared to previously prepared PU electrospun scaffolds, fibrin scaffolds and amniotic membrane scaffold. Significant difference of cell adhesion was evident between the natural and synthetic scaffolds, as for the electrospun scaffolds the significance between, as well as for the NaOH treated samples was less evident. The repetitiveness of the different donor cells fluorescence within the same scaffold material was also present.

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Active immunization against porcine postweaning colidiarrhea (CD) and/or colienterotoxemia (CE) caused by F4+ and/or F18+ enterotoxigenic Escherichia coli (ETEC) is still an unsolved problem. The intestinal microfold (M) cells play role in entry/invasion of intraluminal pathogens (such as ETEC strains), in antigen sampling, and in facilitating induction of immunity to gut infections. Just as ETEC strains can exploit M cells as the portal of entry for infections such as CD and/or CE, their high transcytotic ability make them an attractive target for mucosally delivered vaccines, adjuvants and therapeutics. The objective of our study was to evaluate the effects of levamisole-adjuvanted vaccine candidate F4ac+ and F18ac+non-ETEC strains on incidence/fequency of ileal M cells and up-regulation of antigen delivery by de novo formation of these cells in weaned pigs. Conventionally reared 4-weeks-old pigs were divided into three groups of which two were parenterally and orally immunized with levamisole (at Days -2, -1 and 0) in combination with either vaccine candidate non-ETEC strain (at Day 0), respectively. The third group of pigs received saline as a placebo. Challenge was performed (at Day 7) with F4ac+ ETEC strain, and the pigs were euthanatized (at Day 13) and sampled for immunohistology. Distribution patterns of cytokeratin 18 positive M cells revealed that they are rather solitary interspersed between enterocytes than as small clusters, and most of them were found to be located at the apex of villi in the ileum of 6-weeks-old pigs. Morphometric quantification of M cells in the ileal mucosa showed that levamisole-pretreated F18ac+non-ETEC-immunized and challenged pigs had significantly increased numbers (p