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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.

Cultivation of limbal stem cells (LSC) 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. Limbal cells were obtained from ocular biopsies cultivated on a feeder-layer made from 3T3 cells. This cell therapy was classified as advanced therapy medicinal products (ATMPs).In the Laboratory of Tissue Engineering in microbiological cabinet (which is clean room class A) were we prepared limbal stem cell grafts. With methods of flow cytometry it is possible to detect anti-feeder layer positive cells, stem cells and differentiation cells.Haematoxylin and eosin stained histological images showing firstly cell morphology and secondly stratification of limbal grafts. Immunohistochemistry analysis detected positive staining of stem cells (p63α), Ki67 and CK3. Live-cell imaging of human LSC were obtained in collaboration with Ruđer Bošković Institute, Zagreb. The cell forms a spindle, a molecular machine responsible for equal distribution of chromosomes between the daughter cells. It is recommended to use reagents, growth medium and water certified to contain endotoxin in minimal concentration. Cultivated limbal epithelial transplantation (CLET) has shown to be an effective therapy for LSCD, with clinical trials reporting an average success rate of 70 to 80%

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.

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.

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.

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.

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

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.