1. Razvoj in vitro modela kožeMaja Sever, Dominik Škrinjar, 2024, final research report Abstract: Tridimenzionalni (3D) biotiskani modeli kože se vse bolj uveljavljajo v predkliničnih in kliničnih raziskavah kot alternativa testiranju na živalih. Ključno za uspeh teh raziskav je zagotavljanje ponovljivosti in primerljivosti rezultatov, kar zahteva razvoj zanesljivih hidrogelov, ki omogočajo stabilno vključitev celic v 3D biotisk. Eden največjih izzivov pri tem je zagotavljanje konsistentnosti lastnosti hidrogela, ki je temeljni material za izdelavo 3D tiskanega in vitro modela kože, potrebnega za različne faze raziskav. Namen te raziskave je bil preučiti možnost priprave večje serije hidrogela ter raziskati načine njihovega dolgoročnega shranjevanja, s poudarkom na tehnikah zamrzovanja, z namenom ohranitve njihovih fizikalno-kemijskih lastnosti. Tako bi omogočili uporabo enotne serije hidrogela skozi celoten razvoj in vitro modela kože, kar bi zmanjšalo variacije, ki nastajajo pri posameznih pripravah hidrogelov za različne raziskovalne metode. Osredotočili smo se na vpliv temperature (-20 °C in -80 °C) ter trajanja shranjevanja (7 in 29 dni) na ključne lastnosti hidrogelov, kot so viskoznost, poroznost, sposobnost nabrekanja, degradacija in mehanske lastnosti po odmrznitvi. Potrditev teh hipotez bi prispevala k večji ponovljivosti in natančnosti 3D biotiskljivih in vitro modelov kože. Keywords: in vitro model kože, 3D biotiskanje, hidrogel, predkinična in klinična raziskava Published in DKUM: 24.01.2025; Views: 0; Downloads: 3
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2. Mesenchymal stem cells isolated from paediatric paravertebral adipose tissue show strong osteogenic potentialJan Rožanc, Lidija Gradišnik, Tomaž Velnar, Minja Gregorič, Marko Milojević, Boštjan Vihar, Boris Gole, Uroš Maver, 2022, original scientific article Abstract: Mesenchymal stem cells (MSCs) represent the basis of novel clinical concepts in cellular therapy and tissue regeneration. Therefore, the isolation of MSCs from various tissues has become an important endeavour for stem cell biobanking and the development of regenerative therapies. Paravertebral adipose tissue is readily exposed during spinal procedures in children and could be a viable source of stem cells for therapeutic applications. Here, we describe the first case of MSCs isolated from paravertebral adipose tissue (PV-ADMSCs), obtained during a routine spinal surgery on a child. Using quantitative real-time PCR and flow cytometry, we show that PV-ADMSCs have different levels of stem marker expression compared to the MSCs from other sources while having the highest proliferation rate. Furthermore, we evaluate the multipotency of PV-ADMSCs by the three-lineage (adipogenic, osteogenic and chondrogenic) differentiation and compare it to the multipotency of MSCs from other sources. It was found that the PV-ADMSCs have a strong osteogenic potential in particular. Taken together, our data indicate that PV-ADMSCs meet the criteria for successful cell therapy, defined by the International Society for Cellular Therapy (ISCT), and thus, could provide a source of MSCs that is relatively easy to isolate and expand in culture. Due to their strong osteogenic potential, these cells provide a promising basis, especially for orthopaedic applications. Keywords: mesenchymal stem cells, paediatric surgery, paravertebral adipose tissue, regenerative medicine, stem marker expression, osteogenic potential, differentiation Published in DKUM: 16.12.2024; Views: 0; Downloads: 5
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3. Investigating the viability of epithelial cells on polymer based thin-filmsBoštjan Vihar, Jan Rožanc, Boštjan Krajnc, Lidija Gradišnik, Marko Milojević, Laura Činč Ćurić, Uroš Maver, 2021, original scientific article Abstract: The development of novel polymer-based materials opens up possibilities for several novel applications, such as advanced wound dressings, bioinks for 3D biofabrication, drug delivery systems, etc. The aim of this study was to evaluate the viability of vascular and intestinal epithelial cells on different polymers as a selection procedure for more advanced cell-polymer applications. In addition, possible correlations between increased cell viability and material properties were investigated. Twelve polymers were selected, and thin films were prepared by dissolution and spin coating on silicon wafers. The prepared thin films were structurally characterized by Fourier transform infrared spectroscopy, atomic force microscopy, and goniometry. Their biocompatibility was determined using two epithelial cell lines (human umbilical vein endothelial cells and human intestinal epithelial cells), assessing the metabolic activity, cell density, and morphology. The tested cell lines showed different preferences regarding the culture substrate. No clear correlation was found between viability and individual substrate characteristics, suggesting that complex synergistic effects may play an important role in substrate design. These results show that a systematic approach is required to compare the biocompatibility of simple cell culture substrates as well as more complex applications (e.g., bioinks). Keywords: HUIEC, HUVEC, morphology, polymers, thin films, viability Published in DKUM: 18.10.2024; Views: 0; Downloads: 1
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4. Advanced bio-based polymers for astrocyte cell modelsLidija Gradišnik, Roman Bošnjak, Tina Maver, Tomaž Velnar, 2021, review article Abstract: The development of in vitro neural tissue analogs is of great interest for many biomedical engineering applications, including the tissue engineering of neural interfaces, treatment of neurodegenerative diseases, and in vitro evaluation of cell-material interactions. Since astrocytes play a crucial role in the regenerative processes of the central nervous system, the development of biomaterials that interact favorably with astrocytes is of great research interest. The sources of human astrocytes, suitable natural biomaterials, guidance scaffolds, and ligand patterned surfaces are discussed in the article. New findings in this field are essential for the future treatment of spinal cord and brain injuries. Keywords: astrocytes, hydrogels, tissue engineering Published in DKUM: 14.08.2024; Views: 118; Downloads: 7
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5. Novel methacrylate-based multilayer nanofilms with incorporated FePt-based nanoparticles and the anticancer drug 5-fluorouracil for skin cancer treatmentKristijan Skok, Tanja Zidarič, Kristjan Orthaber, Matevž Pristovnik, Nina Kostevšek, Kristina Žužek Rožman, Sašo Šturm, Lidija Gradišnik, Uroš Maver, Tina Maver, 2022, original scientific article Abstract: Despite medical advances, skin-associated disorders continue to pose a unique challenge to physicians worldwide. Skin cancer is one of the most common forms of cancer, with more than one million new cases reported each year. Currently, surgical excision is its primary treatment; however, this can be impractical or even contradictory in certain situations. An interesting potential alternative could lie in topical treatment solutions. The goal of our study was to develop novel multilayer nanofilms consisting of a combination of polyhydroxyethyl methacrylate (PHEMA), polyhydroxypropyl methacrylate (PHPMA), sodium deoxycholate (NaDOC) with incorporated superparamagnetic iron–platinum nanoparticles (FePt NPs), and the potent anticancer drug (5-fluorouracil), for theranostic skin cancer treatment. All multilayer systems were prepared by spin-coating and characterised by atomic force microscopy, infrared spectroscopy, and contact angle measurement. The magnetic properties of the incorporated FePt NPs were evaluated using magnetisation measurement, while their size was determined using transmission electron microscopy (TEM). Drug release performance was tested in vitro, and formulation safety was evaluated on human-skin-derived fibroblasts. Finally, the efficacy for skin cancer treatment was tested on our own basal-cell carcinoma cell line. Keywords: nanomaterials, bimodal therapy, topical skin treatmen, magnetic nanoparticles, thin films, skin cancer, methacrylates Published in DKUM: 04.07.2024; Views: 98; Downloads: 16
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6. Multilayer methacrylate-based wound dressing as a therapeutic tool for targeted pain reliefTanja Zidarič, Kristijan Skok, Kristjan Orthaber, Matevž Pristovnik, Lidija Gradišnik, Tina Maver, Uroš Maver, 2023, original scientific article Abstract: This study presents an innovative wound dressing system that offers a highly effective therapeutic solution for treating painful wounds. By incorporating the widely used non-steroidal anti-inflammatory drug diclofenac, we have created an active wound dressing that can provide targeted pain relief with ease. The drug was embedded within a biocompatible matrix composed of polyhydroxyethyl methacrylate and polyhydroxypropyl methacrylate. The multilayer structure of the dressing, which allows for sustained drug release and an exact application, was achieved through the layer-by-layer coating technique and the inclusion of superparamagnetic iron platinum nanoparticles. The multilayered dressings’ physicochemical, structural, and morphological properties were characterised using various methods. The synergistic effect of the incorporated drug molecules and superparamagnetic nanoparticles on the surface roughness and release kinetics resulted in controlled drug release. In addition, the proposed multilayer wound dressings were found to be biocompatible with human skin fibroblasts. Our findings suggest that the developed wound dressing system can contribute to tailored therapeutic strategies for local pain relief. Keywords: wound dressings, pain relief, superparamagnetic nanoparticles, methacrylate Published in DKUM: 19.04.2024; Views: 485; Downloads: 468
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7. Efficient and Green Isolation of Keratin from Poultry Feathers by Subcritical WaterMojca Škerget, Maja Čolnik, Lidija Fras Zemljič, Lidija Gradišnik, Tanja Živković Semren, Blanka Tariba Lovaković, Uroš Maver, 2023, original scientific article Abstract: The isolation of keratin from poultry feathers using subcritical water was studied in a batch reactor at temperatures (120–250 °C) and reaction times (5–75 min). The hydrolyzed product was characterized by FTIR and elemental analysis, while the molecular weight of the isolated product was determined by SDS-PAGE electrophoresis. To determine whether disulfide bond cleavage was followed by depolymerization of protein molecules to amino acids, the concentration of 27 amino acids in the hydrolysate was analyzed by GC/MS. The optimal operating parameters for obtaining a high molecular weight protein hydrolysate from poultry feathers were 180 °C and 60 min. The molecular weight of the protein hydrolysate obtained under optimal conditions ranged from 4.5 to 12 kDa, and the content of amino acids in the dried product was low (2.53% w/w). Elemental and FTIR analyses of unprocessed feathers and dried hydrolysate obtained under optimal conditions showed no significant differences in protein content and structure. Obtained hydrolysate is a colloidal solution with a tendency for particle agglomeration. Finally, a positive influence on skin fibroblast viability was observed for the hydrolysate obtained under optimal processing conditions for concentrations below 6.25 mg/mL, which makes the product interesting for various biomedical applications. Keywords: poultry feathers, subcritical water hydrolysis, keratin, physico-chemical characterization, cytotoxicity Published in DKUM: 28.11.2023; Views: 456; Downloads: 157
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8. Amoxicillin doped hyaluronic acid/fucoidan multifunctional coatings for medical grade stainless steel orthopedic implantsMatej Bračič, Sanja Potrč, Matjaž Finšgar, Lidija Gradišnik, Uroš Maver, Hanna Budasheva, Dorota Korte, Mladen Franko, Lidija Fras Zemljič, 2023, original scientific article Abstract: This work investigated the potential of amoxicillin-doped hyaluronic acid/fucoidan multifunctional coatings on medical grade stainless steel as biocompatible, osteointegration enhancing, antimicrobial, and bacterial biofilm inhibiting coatings for orthopedic implants. The coatings were prepared by layer-by-layer spin coating and confirmed by optical contact angle goniometry and infrared spectroscopy. Time-of-flight secondary ion mass spectrometry showed a homogeneous distribution of the individual layers. In contrast, thermal diffusivity, and thermal conductivity measurements by photothermal beam deflection spectrometry showed diffusion of the amoxicillin into the hyaluronic acid and fucoidan layers. In vitro release of amoxicillin showed complete release within one hour, as reflected by the formation of inhibition zones against Staphylococcus aureus and Escherichia coli. Synergistic effects were observed between the hyaluronic acid and amoxicillin in inhibiting S.Aureus biofilm, and between the fucoidan and amoxicillin in improving the antioxidant properties by an ABTS radical scavenging assay. Biocompatibility was determined with human osteoblasts, confirming the potential of such multifunc- tional coatings to enhance the bioactivity of steel-based orthop edic implants. Keywords: orthopedic implant, medical grade stainless steel, amoxicillin, hyaluronic acid, fucoidan, multifunctional coating Published in DKUM: 18.08.2023; Views: 588; Downloads: 108
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9. Biomaterials and host versus graft response : a short reviewTomaž Velnar, Gorazd Bunc, Robert Klobucar, Lidija Gradišnik, 2016, review article Abstract: Biomaterials and biotechnology are increasing becoming an important area in modern medicine. The main aim in this area is the development of materials, which are biocompatible to normal tissue. Tissue-implant interactions with molecular, biological and cellular characteristics at the implant-tissue interface are important for the use and development of implants. Implantation may cause an inflammatory and immune response in tissue, foreign body reaction, systemic toxicity and imminent infection. Tissue-implant interactions determine the implant life-period. The aims of the study are to consider the biological response to implants. Biomaterials and host reactions to implants and their mechanisms are also briefly discussed. Keywords: host versus graft disease, GVHD, biomaterial, wound healing, transplant, tissue, prosthetic, implants, biological response, complications Published in DKUM: 03.08.2017; Views: 1466; Downloads: 211
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10. Isolation and characterization of human articular chondrocytes from surgical waste after total knee arthroplasty (TKA)Jakob Naranđa, Lidija Gradišnik, Mario Gorenjak, Matjaž Vogrin, Uroš Maver, 2017, original scientific article Abstract: BACKGROUND: Cartilage tissue engineering is a fast-evolving field of biomedical engineering, in which the chondrocytes represent the most commonly used cell type. Since research in tissue engineering always consumes a lot of cells, simple and cheap isolation methods could form a powerful basis to boost such studies and enable their faster progress to the clinics. Isolated chondrocytes can be used for autologous chondrocyte implantation in cartilage repair, and are the base for valuable models to investigate cartilage phenotype preservation, as well as enable studies of molecular features, nature and scales of cellular responses to alterations in the cartilage tissue.
METHODS: Isolation and consequent cultivation of primary human adult articular chondrocytes from the surgical waste obtained during total knee arthroplasty (TKA) was performed. To evaluate the chondrogenic potential of the isolated cells, gene expression of collagen type 2 (COL2), collagen 1 (COL1) and aggrecan (ACAN) was evaluated. Immunocytochemical staining of all mentioned proteins was performed to evaluate chondrocyte specific production.
RESULTS: Cartilage specific gene expression of COL2 and ACAN has been shown that the proposed protocol leads to isolation of cells with a high chondrogenic potential, possibly even specific phenotype preservation up to the second passage. COL1 expression has confirmed the tendency of the isolated cells dedifferentiation into a fibroblast-like phenotype already in the second passage, which confirms previous findings that higher passages should be used with care in cartilage tissue engineering. To evaluate the effectiveness of our approach, immunocytochemical staining of the evaluated chondrocyte specific products was performed as well.
DISCUSSION: In this study, we developed a protocol for isolation and consequent cultivation of primary human adult articular chondrocytes with the desired phenotype from the surgical waste obtained during TKA. TKA is a common and very frequently performed orthopaedic surgery during which both femoral condyles are removed. The latter present the ideal source for a simple and relatively cheap isolation of chondrocytes as was confirmed in our study. Keywords: aggrecan, collagen 2, gene expression, human articular chondrocytes, isolation protocol, phenotype preservation, TKA, total knee arthroplasty Published in DKUM: 02.08.2017; Views: 1730; Downloads: 656
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