BACKGROUND/AIM: This study investigated the therapeutic potential of lipophosphonoxin (LPPO), an antibacterial agent, loaded into polycaprolactone nanofiber dressings (NANO-LPPO) for full-thickness wound healing. Using a porcine model, we aimed to assess the impact of areal weight of the dressing (10, 20 and 30 g/m2) on wound-healing outcomes and validate findings from previous murine studies. MATERIALS AND METHODS: Full-thickness wounds were created on porcine skin and treated with the NANO-LPPO dressings of differing thickness. Positive control (Aquacel Ag+) and standard control (Jelonet) groups were included for comparison. Wound-healing progression was evaluated macroscopically and on the histological level. RESULTS: Macroscopic observations indicated no signs of infection in any group, with wounds covered by scabs by day 14. Thicker dressings (areal weights of 30 and 20 g/m2) demonstrated superior performance in promoting the formation of granulation tissue and healing compared to the thinner version (areal weight of 10 g/m2). LPPO-loading enhanced scaffold wettability and biodegradability without impairing healing outcomes. Both control groups exhibited similar healing characteristics. CONCLUSION: The findings underscore the importance of optimizing dressing thickness for effective wound healing. NANO-LPPO dressings exhibit translational potential as a therapeutic option for full-thickness wounds, warranting further preclinical and regulatory evaluation to support clinical application.
- MeSH
- antibakteriální látky farmakologie aplikace a dávkování chemie MeSH
- hojení ran * účinky léků MeSH
- kůže účinky léků patologie MeSH
- lipoxiny * chemie farmakologie aplikace a dávkování MeSH
- modely nemocí na zvířatech MeSH
- nanovlákna * chemie MeSH
- obvazy * MeSH
- polyestery * chemie MeSH
- prasata MeSH
- zvířata MeSH
- Check Tag
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
PURPOSE: This study aimed to evaluate early-phase safety of subretinal application of AAVanc80.CAG.USH1Ca1 (OT_USH_101) in wild-type (WT) pigs, examining the effects of a vehicle control, low dose, and high dose. METHODS: Twelve WT pigs (24 eyes) were divided into three groups: four pigs each received bilateral subretinal injections of either vehicle, low dose (3.3 × 1010 vector genomes [vg] per eye), or high dose (1.0 × 1011 vg per eye). Total retinal thickness (TRT) was evaluated using optical coherence tomography and retinal function was assessed with full-field electroretinography (ff-ERG) at baseline and two months post-surgery. After necropsy, retinal changes were examined through histopathology, and human USH1C_a1/harmonin expression was assessed by quantitative PCR (qPCR) and Western blotting. RESULTS: OT_USH_101 led to high USH1C_a1 expression in WT pig retinas without significant TRT changes two months after subretinal injection. The qPCR revealed expression of the human USH1C_a1 transgene delivered by the adeno-associated virus vector. TRT changes were minimal across groups: vehicle (256 ± 21 to 243 ± 18 μm; P = 0.108), low dose (251 ± 32 to 258 ± 30 μm; P = 0.076), and high dose (242 ± 24 to 259 ± 28 μm; P = 0.590). The ff-ERG showed no significant changes in rod or cone responses. Histopathology indicated no severe retinal adverse effects in the vehicle and low dose groups. CONCLUSIONS: Early-phase clinical imaging, electrophysiology, and histopathological assessments indicated that subretinal administration of OT_USH_101 was well tolerated in the low-dose treatment arm. OT_USH_101 treatment resulted in high expression of human USH1C_a1. Although histopathological changes were not severe, more frequent changes were observed in the high-dose group.
- MeSH
- cytoskeletální proteiny genetika MeSH
- Dependovirus genetika MeSH
- elektroretinografie * MeSH
- genetická terapie metody MeSH
- genetické vektory * MeSH
- injekce nitrooční * MeSH
- kvantitativní polymerázová řetězová reakce MeSH
- lidé MeSH
- modely nemocí na zvířatech MeSH
- optická koherentní tomografie * MeSH
- prasata MeSH
- proteiny buněčného cyklu genetika MeSH
- regulace genové exprese MeSH
- retina * metabolismus patologie MeSH
- transgeny * MeSH
- western blotting MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
PURPOSE: Subretinal (SR) injection in porcine models is a promising avenue for preclinical evaluation of cell and gene therapies. Targeting of the subretinal fluid compartment (bleb) is critical to the procedure, especially if treatment of the cone-rich area centralis is required (i.e., visual streak [VS] in pigs). To our knowledge, this study is the first to investigate the influence of injection site placement on VS involvement in the pig eye. METHODS: We performed 23-gauge pars plana vitrectomy followed by SR injection in 41 eyes of 21 animals (Sus scrofa domesticus). In 27 eyes (65.9%), the injection site was placed superior to the VS, and in 14 eyes (34.1%) it was placed inferior to it. Using intraoperative imaging, blebs were classified based on their propagation behavior relative to the VS. RESULTS: In 79% of cases, blebs from inferior injection sites developed away from the VS, exhibiting a mean ± SEM vertical anisotropy (AP) of 0.67 ± 0.11. In contrast, blebs from superior injection sites tended to develop toward the VS with an AP of 1.27 ± 0.18 (P = 0.0070). Blebs developed away from the VS in only 41% of injections (P = 0.0212). Inferior blebs were orientated close to 0° (horizontal), whereas superior blebs displayed varied orientations with a mean angle of 56° (P = 0.0008). CONCLUSIONS: Bleb propagation was anisotropic (i.e., directionally biased) and dependent on injection site placement. Superior injection sites led to superior VS detachment. Morphological analysis suggested increased adhesion forces at the VS and superior vascular arcades. This study will aid the planning of surgeries for targeted subretinal delivery in pig models.
Technologie indukované pluripotence, která umožňuje přípravu prakticky jakéhokoliv buněčného modelu, potřebného pro výzkum vybraného onemocnění, se stává významným nástrojem v boji proti pandemii koronaviru SARS‐CoV-2 a případně i jiným formám pandemií, které se mohou s velkou pravděpodobností objevit v blízké, či vzdálené budoucnosti. Je výhodné, pokud lidstvo v předstihu disponuje arzenálem vhodných modelových systémů, které je možné v případě potřeby nasadit do boje a využít i pro vývoj nových terapeutických a diagnostických strategií. S ohledem na aktuální epidemickou situaci se tak významným způsobem může zkrátit čas, nezbytný pro nalezení vhodného léčebného postupu či zavedení spolehlivého diagnostického setu. Indukované pluripotentní kmenové buňky mají zároveň potenciál použití při regeneraci tkáně, poškozené samotnou infekcí. Jejich značnou výhodou na poli regenerativní medicíny je imunologicky atraktivní možnost využití tělu vlastních, tzv. autologních buněk, bez nutnosti nasazení imunosupresivní terapie po transplantaci.
Technology of induced pluripotency allows the preparation of any cell model for research of any selected disease and becomes the important tool also in the field of SARS‐CoV-2 coronavirus pandemic and other forms of pandemics that can appear in near or far future. It is beneficial for humankind to have an arsenal of useful model systems that can be immediately used in development of novel therapeutic or diagnostic strategies whenever needed. The possible use of newly developed therapy or diagnostic set can be greatly accelerated during pandemic situation. Induced pluripotent stem cells that can be prepared by technology of induced pluripotency also have great potential in tissue regeneration for tissues damaged by coronavirus infection. Immunologically very attractive is the fact that induced pluripotent stem cells can be applied (transplanted) without the use of aggressive immunosuppressive treatment.
- MeSH
- biologické modely MeSH
- COVID-19 terapie MeSH
- indukované pluripotentní kmenové buňky * MeSH
- lidé MeSH
- organoidy MeSH
- techniky in vitro * metody MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- práce podpořená grantem MeSH
- přehledy MeSH
Here, we present newly derived in vitro model for modeling Duchenne muscular dystrophy. Our new cell line was derived by reprogramming of peripheral blood mononuclear cells (isolated from blood from pediatric patient) with Sendai virus encoding Yamanaka factors. Derived iPS cells are capable to differentiate in vitro into three germ layers as verified by immunocytochemistry. When differentiated in special medium, our iPSc formed spontaneously beating cardiomyocytes. As cardiomyopathy is the main clinical complication in patients with Duchenne muscular dystrophy, the cell line bearing the dystrophin gene mutation might be of interest to the research community.
One of the challenges in clinical translation of cell-replacement therapies is the definition of optimal cell generation and storage/recovery protocols which would permit a rapid preparation of cell-treatment products for patient administration. Besides, the availability of injection devices that are simple to use is critical for potential future dissemination of any spinally targeted cell-replacement therapy into general medical practice. Here, we compared the engraftment properties of established human-induced pluripotent stem cells (hiPSCs)-derived neural precursor cell (NPCs) line once cells were harvested fresh from the cell culture or previously frozen and then grafted into striata or spinal cord of the immunodeficient rat. A newly developed human spinal injection device equipped with a spinal cord pulsation-cancelation magnetic needle was also tested for its safety in an adult immunosuppressed pig. Previously frozen NPCs showed similar post-grafting survival and differentiation profile as was seen for freshly harvested cells. Testing of human injection device showed acceptable safety with no detectable surgical procedure or spinal NPCs injection-related side effects.
- MeSH
- buněčná diferenciace fyziologie MeSH
- dospělí MeSH
- genetické vektory genetika MeSH
- indukované pluripotentní kmenové buňky * fyziologie transplantace MeSH
- krysa rodu rattus MeSH
- lidé MeSH
- mícha MeSH
- mozek MeSH
- nervové kmenové buňky * fyziologie transplantace MeSH
- odběr biologického vzorku metody MeSH
- odběr tkání a orgánů metody MeSH
- prasata MeSH
- přeprogramování buněk * genetika fyziologie MeSH
- přežívání štěpu fyziologie MeSH
- spinální injekce * škodlivé účinky přístrojové vybavení metody MeSH
- transplantace kmenových buněk * škodlivé účinky přístrojové vybavení metody MeSH
- virus Sendai MeSH
- výsledek terapie MeSH
- zvířata MeSH
- Check Tag
- dospělí MeSH
- krysa rodu rattus MeSH
- lidé MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
BACKGROUND: Nanofiber wound dressings remain the domain of in vitro studies. The purpose of our study was to verify the benefits of chitosan (CTS) and polylactide (PLA)-based nanofiber wound dressings on a porcine model of a naturally contaminated standardized wound and compare them with the conventional dressings, i.e., gauze and Inadine. MATERIAL AND METHODS: The study group included 32 pigs randomized into four homogeneous groups according to the wound dressing type. Standardized wounds were created on their backs, and wound dressings were regularly changed. We evaluated difficulty of handling individual dressing materials and macroscopic appearance of the wounds. Wound swabs were taken for bacteriological examination. Blood samples were obtained to determine blood count values and serum levels of acute phase proteins (serum amyloid A, C-reactive protein, and haptoglobin). The crucial point of the study was histological analysis. Microscopic evaluation was focused on the defect depth and tissue reactions, including formation of the fibrin exudate with neutrophil granulocytes, the layer of granulation and cellular connective tissue, and the reepithelialization. Statistical analysis was performed by using SPSS software. The analysis was based on the Kruskal-Wallis H test and Mann-Whitney U test followed by Bonferroni correction. Significance was set at P < .05. RESULTS: Macroscopic examination did not show any difference in wound healing among the groups. However, evaluation of histological findings demonstrated that PLA-based nanofiber dressing accelerated the proliferative (P = .025) and reepithelialization (P < .001) healing phases, while chitosan-based nanofiber dressing potentiated and accelerated the inflammatory phase (P = .006). No statistically significant changes were observed in the blood count or acute inflammatory phase proteins during the trial. Different dynamics were noted in serum amyloid A values in the group treated with PLA-based nanofiber dressing (P = .006). CONCLUSION: Based on the microscopic examination, we have documented a positive effect of nanofiber wound dressings on acceleration of individual phases of the healing process. Nanofiber wound dressings have a potential to become in future part of the common wound care practice.
The critical requirements in developing clinical-grade human-induced pluripotent stem cells-derived neural precursors (hiPSCs-NPCs) are defined by expandability, genetic stability, predictable in vivo post-grafting differentiation, and acceptable safety profile. Here, we report on the use of manual-selection protocol for generating expandable and stable human NPCs from induced pluripotent stem cells. The hiPSCs were generated by the reprogramming of peripheral blood mononuclear cells with Sendai-virus (SeV) vector encoding Yamanaka factors. After induction of neural rosettes, morphologically defined NPC colonies were manually harvested, re-plated, and expanded for up to 20 passages. Established NPCs showed normal karyotype, expression of typical NPCs markers at the proliferative stage, and ability to generate functional, calcium oscillating GABAergic or glutamatergic neurons after in vitro differentiation. Grafted NPCs into the striatum or spinal cord of immunodeficient rats showed progressive maturation and expression of early and late human-specific neuronal and glial markers at 2 or 6 months post-grafting. No tumor formation was seen in NPCs-grafted brain or spinal cord samples. These data demonstrate the effective use of in vitro manual-selection protocol to generate safe and expandable NPCs from hiPSCs cells. This protocol has the potential to be used to generate GMP (Good Manufacturing Practice)-grade NPCs from hiPSCs for future clinical use.
- MeSH
- buněčná diferenciace MeSH
- indukované pluripotentní kmenové buňky * MeSH
- krysa rodu rattus MeSH
- leukocyty mononukleární MeSH
- lidé MeSH
- nervové kmenové buňky * MeSH
- neurony metabolismus MeSH
- virus Sendai genetika MeSH
- zvířata MeSH
- Check Tag
- krysa rodu rattus MeSH
- lidé MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
We present here a new iPS cell line for modeling sporadic form of ALS. Cell line was generated by reprogramming skin fibroblasts isolated with explant culture technology from skin biopsy, donated by ALS patient. For reprogramming, polycistronic self-replicating RNA vector was used and derived iPS cells were characterized by immunocytochemistry and FACS (pluripotent factors expression), karyotyping, STR fingerprinting analysis and in vitro differentiation assay. New cell line showed normal (46, XY) karyotype and differentiated in vitro into cells from three germ layers. STR analysis proved the origin and originality of the cell line.
- MeSH
- amyotrofická laterální skleróza * patologie MeSH
- buněčná diferenciace MeSH
- buněčné linie MeSH
- fibroblasty metabolismus MeSH
- indukované pluripotentní kmenové buňky * metabolismus MeSH
- lidé MeSH
- technologie MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
