Treatment of Total Limbal Stem Cell Deficiency With Autologous Ex Vivo Cultivated Limbal Epithelial Stem Cell Graft

Limbal epithelial stem cell (LSC) deficiency is severe disease of the anterior eye surface, causing corneal opacification and significant vision loss in the affected patients. Treatment of such patients by corneal graft is ineffective due to the fact that corneal donor tissue cannot survive in the eye without LSC. One of possible treatment options is to provide LSC from the other, unaffected eye, and to perform corneal graft later on, as a second procedure. In this paper, we have evaluated the visual outcome and clinical improvement in three eyes receiving ex vivo cultivated limbal epithelial stem cell graft to treat their limbal stem cell deficiency (LSCD) caused by corneal burn. We aimed at determining whether this treatment alone might provide sufficient visual improvement to avoid corneal grafting as a second surgery

De novo diferencijacija M stanica crijevnih resica u odbijene prasadi imunizirane pokusnim cjepnim F4ac+ ili F18ac+ neenterotoksigenim sojevima bakterije Escherichia coli s levamisolom kao adjuvansom.

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 a role in the entry/invasion of intraluminal pathogens (such as ETEC strains), in antigen sampling, and in facilitating the 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 makes 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/frequency of ileal M cells and up-regulation of antigen delivery by de novo formation of these cells in weaned pigs. Conventionally reared 4-week-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 the 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 interspersed between enterocytes than as small clusters, and most of them were found to be located at the apex of the villi in the ileum of 6-weeks-old-pigs. Morphometric quantififi cation of M cells in the ileal mucosa showed that levamisole-pretreated F18ac+non-ETEC-immunized and challenged pigs had significantly increased numbers (P<0.01) of ileal M cells as compared to the values obtained in the control non-primed and challenged pigs. The proportion of these cells in this group of pigs was increased by 145%. In the levamisole-pretreated F4ac+ non-ETEC-immunized and challenged pigs only a slightly increased (for 7%) proportion of M cells was recorded. However, this increase was not significantly different from the numerical values obtained for control pigs. Our finding that levamisole-adjuvanted F18ac+non-ETEC vaccine may affect de novo differentiation of antigen-sampling M cells by increasing their number in the ileum, indicated that the vaccine probably utilizes these cells as a target for entry/delivery to the nearby lymphocytes and induces protective immunity against CE. On the other hand, the failure of levamisole-adjuvanted F4ac+non-ETEC vaccine to produce a similar effect on M cells remains to be elucidated.Aktivna imunizacija odbijene prasadi protiv kolidijareje (KD) i/ili kolienterotoksemije (KE) uzrokovane F4ac+ i/ili F18ac+ enterotoksigenim sojevima bakterije Escherichia coli (ETEC) još je uvijek neriješen problem. Crijevne mikronaborane (M) stanice imaju funkciju pri ulasku/invaziji intraluminalnih patogena (kao što su ETEC sojevi), unosu uzoraka antigena i pogodovanju tvorbi imunosti na probavne infekcije. Činjenica da ETEC sojevi rabe M stanice kao ulazna vrata za uzročnike infekcija, kao što su KD i/ili KE, a njihova velika sposobnost transcitoze čini ih ujedno pogodnim stanicama za unos mukoznih cjepiva, adjuvanata i lijekova. Cilj ovoga rada bilo je vrednovanje učinaka pokusnih cjepnih sojeva F4ac+ i F18ac+ ne-ETEC, s levamisolom kao adjuvansom, na pojavnost i brojnost M stanica u ileumu, te na poticanje unosa antigena nakon de novo tvorbe tih stanica u odbijene prasadi. Prasad iz uobičajenoga uzgoja, u dobi od 4 tjedna, bila je razvrstana u tri skupine od kojih su dvije parenteralno/oralno imunizirane levamisolom (-2., -1. i 0. dana pokusa) u kombinaciji s jednim od dva pokusna vakcinalna ne-ETEC soja (0. dana pokusa). Treća je skupina primila fiziološku otopinu kao placebo. Izazivačka je infekcija provedena s F4ac+ ETEC sojem (7. dana pokusa), a prasad je usmrćena (13. dana pokusa) radi uzimanja uzoraka za imunohistologiju. Distribucijski obrasci M stanica, pozitivnih na biljeg za citokeratin 18, pokazuju da su pretežito raspršene između enterocita, a manje ih je u malim nakupinama, te da se većina tih stanica nalazi pri vrhu resica ileuma prasadi u dobi od 6 tjedana. Morfometrijska kvantifikacija M stanica u sluznici ileuma pokazuje da prasad koja je prethodno dobivala levamisol i imunizirana F18ac+ ne- ETEC sojem ima značajno veći broj (P<0,01) M stanica u usporedbi s vrijednostima dobivenima u kontrolne neimunizirane prasadi. Udjel M stanica u te prasadi bio je povećan za 145%. U skupini prasadi prethodno obrađene levamisolom i imuniziranih F4ac+ ne-ETEC sojem zabilježen je samo blagi porast (za 7%) udjela M stanica. Međutim, taj porast nije bio statistički značajno različit od vrijednosti dobivenih u kontrolne prasadi. Naš nalaz da F18ac+ ne-ETEC vakcina s levamisolom kao adjuvansom može pospješiti de novo diferencijaciju M stanica time što povećava njihovu brojnost u ileumu, upućuje na povećanu sposobnost tih stanica da unose pokusni vakcinalni soj i tako dostavljaju imunogen do obližnjih limfocita što potiče zaštitnu imunost protiv KE. Međutim, trebalo bi objasniti izostanak sličnog učinka na M stanice u prasadi koja je primila F4ac+ ne-ETEC vakcinu s levamisolom kao adjuvansom

Electrospun Polycaprolactone for Controlled Drug Delivery

Tkivno inženjerstvo dobra je alternativa za razvoj tkiva ili potencijalnih organa iz pacijentova vlastitog staničnog materijala, a kako bi se smanjio problem nedostatka organa za transplantaciju. Elektroispredeni materijali su dobri kandidati kod primjene u biomedicini tj. kao nosači za uzgoj tkivnih stanica. Kod regeneracije tkiva mogu dodatno prenositi lijekove kontrolirano prema terapiji. U ovom je radu istraživano kontrolirano otpuštanje antibiotika Cefuroxima (CFU) koji se upotrebljava u terapiji okularnog tkiva. Elektroispredeni su polikaprolaktonski nosači uz dodatak CFU-a u udjelima od 1, 2, 5 i 10 mas %. Uspješna kapsulacija antibiotika potvrđena je pojavom novih karakterističnih pikova u FTIR spektrima elektroispredenih mješavina. Dodatkom antibiotika i povećanjem njegove koncentracije dobivaju se vlakna homogenijeg izgleda s manjim brojem deformacija po duljini vlakna. UV-VIS spektrofotometrijom praćeno je vrijeme otpuštanja antibiotika iz elektroispredenih PCL/CFU nosača. Dobiveno je povećanje apsorbancije antibiotika s vremenom i porastom koncentracije lijeka u nosaču. Ovo djelo je dano na korištenje pod licencom Creative Commons Imenovanje 4.0 međunarodna.Tissue engineering is a good alternative for the development of tissue or potential organs from the patient’s own cell material in order to reduce the problem of organ transplant deficiency. Electrospun materials are good candidates for use in biomedicine, as scaffolds for tissue cells culture. Additionally, these scaffolds can provide controlled drug release in tissue regenerative therapies. In this paper, controlled release of antibiotic Cefuroxim (CFU), which is used for ocular tissue therapy, was investigated. The polycaprolactone scaffolds were prepared by electrospinning with the addition of CFU in the amount of 1, 2, 5, and 10 wt %. The successful antibiotic capsulation was confirmed by the new characteristic peaks appearing in the FTIR spectra of the electrospun blends. With the addition of antibiotic and increase in its concentration, fibres with more uniform morphology and less deformations along the fibres length were obtained. The release of antibiotic from PCL scaffolds was determined by UV–VIS spectrophotometer. Obtained was an increase in absorption of antibiotics with time and with increased drug concentration in the scaffolds. This work is licensed under a Creative Commons Attribution 4.0 International License

Fibrin u tkivnom inženjerstvu kože – proizvodnja i kliničko iskustvo

The purpose of this study was to create a fibrin-based human skin substitute in vitro with epidermal and dermal component and to assess its healing potential in deep partial and full thickness burns. Fibrin scaffolds were prepared from commercial fibrin glue kits. Human fibroblasts were cultured in fibrin gel. Human keratinocytes were seeded on the top of the gel. Viability of cells was determined fluorimetrically. Scanning electron microscope and immunocytochemistry analysis of cultured cells were performed. After hydrosurgical preparation of deep burn necrotic tissue, wound bed was prepared for skin substitutes. Progress of healing was documented using visual estimation and photos. Scanning electron microscope images showed good cell attachment and colony spreading of keratinocytes and fibroblasts on fibrin scaffold. Immunofluorescent staining of cell cultures on fibrin scaffold showed expression of vimentin, a marker of fibroblast cells, cytokeratin 19, a marker of epithelial stem cells, as well as involucrin, a marker of differentiated keratinocytes. Clinical results clearly showed that appearance of the skin did not differ significantly from the areas of transplanted skin using split-thickness skin graft techniques. In conclusion, using these fibrin-cultured autografts on massive full-thickness burn resulted in good healing.Cilj ovoga istraživanja bio je kreirati in vitro nadomjestak ljudske kože s epidermalnim i dermalnim dijelom. Pritom smo koristili fibrinski gel kao nosač stanica. Procijenili smo učinak nadomjestka kože na cijeljenje opeklina drugog i trećeg stupnja. Fibrinski gel načinjen je od komercijalnog kirurškog fibrinskog ljepila. Ljudski fibroblasti kultivirani su u samom fibrinskom nosaču. Ljudski keratinociti zasijani su na gornju površinu nosača. Vijabilnost uzgojenih stanica određena je fluorimetrijski. Nadomjestak kože analiziran je elektronskim skenirajućim mikroskopom. Napravljena je imunocitokemijska analiza kultiviranih stanica. Skenirajući mikroskop pokazao je dobro prianjanje i proliferaciju stanica kože na nosaču. Imunocitokemijska analiza pokazala je prisutnost vimentina, biljega fibroblastnih stanica; citokeratina 19, biljega epitelnih matičnih stanica; te involukrina, biljega diferenciranih keratinocita. Klinička primjena ovih nadomjestaka kože pokazala je slične rezulatate cijeljenja kao i dijelovi rana koji su prekriveni autolognim presatcima kože djelomične debljine. Zaključujemo da nadomjestci kože bazirani na fibrinskom nosaču imaju potencijala u liječenju teških opeklina

Preparation and Characterization of Electrospun PCL/Silk Fibroin Scaffolds

Natural polymer-based scaffolds are generally considered as favourable matrices for the adhesion and growth of cells in tissue repair. One of the most popular materials in this respect is silk fibroin, known for its wide usage in biomedical applications. This work focuses on the development of electrospun scaffolds based on poly(ε-caprolactone) (PCL) and silk fibroin (SF) evaluated regarding the SF effect on their morphology, surface wetting ability, thermal properties, and HaCaT model cell line biocompatibility. The study revealed that the lowest PCL/SF concentration resulted in highest bead-like morphology formation, relatively thick fibers with the presence of random beads in the case of PCL, while uniform and thinner fibers in the case of increasing PCL/SF content scaffolds. The addition of SF reduced the degree of crystallinity in the PCL due to the less organized crystal structure, and decreased its thermal stability. Both SEM and MTT analyses showed cell presence on all scaffolds three days after cell seeding. Although SF improved PCL hydrophilicity, as shown quantitatively by the MTT assay for improved cytocompatibility properties, more structured electrospun PCL/SF scaffold strategies are required. This work is licensed under a Creative Commons Attribution 4.0 International License

Synthetic vs natural scaffolds for human limbal stem cells

Aim To investigate the impact of synthetic electrospun polyurethane (PU) and polycaprolactone (PCL) 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. Methods Human placenta was taken at elective cesarean 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, PU, and PCL nanoscaffolds, with or without prior NaOH treatment. Results Scanning electron microscope photographs of all tested scaffolds showed good colony spreading of seeded limbal cells. There was a significant difference in viability performance 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. Conclusion Natural scaffolds, fibrin and amniotic membrane, showed better cell viability than electrospun scaffolds. On the contrary, high percentages of p63 positive cells obtained on these scaffolds still makes them good candidates for efficient delivery systems for therapeutic purpose

The Reliability of PCL/Anti-VEGF Electrospun Scaffolds to Support Limbal Stem Cells for Corneal Repair

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 confirmed 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

Bioactivity Comparison of Electrospun PCL Mats and Liver Extracellular Matrix as Scaffolds for HepG2 Cells

Cells grown on bioactive matrices have immensely advanced many aspects of biomedical research related to drug delivery and tissue engineering. Our main objective was to perform simple evaluation of the structural and biotic qualities of cell scaffolds made of affordable biomaterials for liver cell line (HepG2) cultivation in vitro. In this work the electrospun matrix made of synthetic polyester poly(ε-caprolactone) (PCL) was compared with the natural protein-based extracellular matrix isolated from porcine liver (ECM). Mechanical and structural analysis showed that ECM was about 12 times less resistant to tensile stress while it had significantly larger pore size and twice smaller water contact angle than PCL. Bioactivity assessment included comparison of cell growth and transfection efficiency on cell-seeded scaffolds. Despite the differences in composition and structure between the two respective matrices, the rate of cell spreading and the percentage of transfected cells on both scaffolds were fairly comparable. These results suggest that in an attempt to produce simple, cell carrying structures that adequately simulate the natural scaffold, one can rely on PCL electrospun mats

Bioactivity Comparison of Electrospun PCL Mats and Liver Extracellular Matrix as Scaffolds for HepG2 Cells

Cells grown on bioactive matrices have immensely advanced many aspects of biomedical research related to drug delivery and tissue engineering. Our main objective was to perform simple evaluation of the structural and biotic qualities of cell scaffolds made of affordable biomaterials for liver cell line (HepG2) cultivation in vitro. In this work the electrospun matrix made of synthetic polyester poly(&epsilon;-caprolactone) (PCL) was compared with the natural protein-based extracellular matrix isolated from porcine liver (ECM). Mechanical and structural analysis showed that ECM was about 12 times less resistant to tensile stress while it had significantly larger pore size and twice smaller water contact angle than PCL. Bioactivity assessment included comparison of cell growth and transfection efficiency on cell-seeded scaffolds. Despite the differences in composition and structure between the two respective matrices, the rate of cell spreading and the percentage of transfected cells on both scaffolds were fairly comparable. These results suggest that in an attempt to produce simple, cell carrying structures that adequately simulate the natural scaffold, one can rely on PCL electrospun mats

Electrospinning of PCL/CEFUROXIM® fibrous scaffolds on 3D printed collectors

In this paper, the effect of different collector geometry on the PCL scaffold architecture and cell viability was investigated. PCL scaffolds with antibiotic CefuroximVR , 15%wt. and 20%wt., were electrospun and characterized by ATR-FTIR, while thermal stability were observed by DSC and TG. PCL/15%CFUVR scaffolds were electrospun on six 3D printed collectors, three ribbed and three mesh geometries, to produce the most favourable structure for cells adhesion and proliferation. SEM study and MTT assay revealed that collectors with the widest slot in both ribbed and mesh geometries exhibited the most favourable structure to enhance cells adhesion. In general, cells spreading on the surface and cells viability proved to be better for the ribbed collectors. The study showed that by controlling the collector geometry and the scaffold three-dimensional structure, it is possible to control cell attachment and differentiation and, consequently, to adjust them for the special tissue demands in the regeneration process