Full recovery from spinal cord injury requires axon regeneration to re-establish motor and sensory pathways. In mammals, the failure of sensory and motor axon regeneration has many causes intrinsic and extrinsic to neurons, amongst which is the lack of adhesion molecules needed to interact with the damaged spinal cord. This study addressed this limitation by expressing the integrin adhesion molecule α9, along with its activator kindlin-1, in sensory neurons via adeno-associated viral (AAV) vectors. This enabled sensory axons to regenerate through spinal cord injuries and extend to the brainstem, restoring sensory pathways, touch sensation and sensory behaviours. One of the integrin ligands in the injured spinal cord is tenascin-C, which serves as a substrate for α9β1 integrin, a key receptor in developmental axon guidance. However, the adult PNS and CNS neurons lack this receptor. Sensory neurons were transduced with α9 integrin (which pairs with endogenous β1 to form a α9β1 tenascin receptor) together with the integrin activator kindlin-1. Regeneration from sensory neurons transduced with α9integrin and kindlin-1 was examined after C4 and after T10 dorsal column lesions with C6,7 and L4,5 sensory ganglia injected with AAV1 vectors. In animals treated with α9 integrin and kindlin-1, sensory axons regenerated through tenascin-C-expressing connective tissue strands and bridges across the lesions and then re-entered the CNS tissue. Many axons regenerated rostrally to the level of the medulla. Axons grew through the dorsal grey matter rather than their normal pathway the dorsal columns. Growth was slow, axons taking 12 weeks to grow from T10 to the medulla, a distance of 4-5 cm. Functional recovery was confirmed through cFos activation in neurons rostral to the injury after nerve stimulation and VGLUT1/2 staining indicating new synapse formation above the lesion. Behavioural recovery was seen in both heat and mechanical sensation, as well as tape removal tests. This approach demonstrates the potential of integrin-based therapies for long distance sensory axon regeneration and functional recovery following thoracic and partial recovery after cervical spinal cord injury.

• MeSH
• axony MeSH
• Dependovirus genetika   MeSH
• genetické vektory MeSH
• krysa rodu rattus MeSH
• modely nemocí na zvířatech MeSH
• myši MeSH
• nervové receptory * metabolismus   fyziologie   patologie   MeSH
• obnova funkce fyziologie   MeSH
• poranění míchy * patologie   patofyziologie   metabolismus   MeSH
• potkani Sprague-Dawley MeSH
• proteiny nervové tkáně metabolismus   genetika   MeSH
• regenerace nervu * fyziologie   MeSH
• tenascin metabolismus   MeSH
• zvířata MeSH
• Check Tag
• krysa rodu rattus MeSH
• myši MeSH
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Research Support, U.S. Gov't, Non-P.H.S. MeSH

BACKGROUND: Cytokine licensing with pro-inflammatory molecules, such as tumour necrosis factor-alpha (TNF-α) and interferon-gamma (IFN-γ), has emerged as a promising strategy to enhance the therapeutic potential of multipotent mesenchymal stromal cells (MSCs). While licensing has demonstrated benefits for immunomodulation, its effects on other key MSC functions, including differentiation and paracrine activity, remain incompletely explored. In this study, we evaluated the transcriptomic, metabolomic, and functional changes induced by short-term TNF-α/IFN-γ priming of Wharton's jelly-derived MSCs (WJ-MSCs). METHODS: WJ-MSCs were expanded and exposed to TNF-α and IFN-γ (10 ng/ml each) for 24 h. Transcriptomic analysis was performed using RNA sequencing to identify differentially expressed genes related to immune modulation and lineage commitment. Metabolomic profiling was conducted using high-resolution mass spectrometry to assess changes in metabolic pathways. Functional assays evaluated the effects of cytokine priming on induced differentiation and growth factor secretion. RESULTS: Cytokine licensing induced notable alterations in gene expression, upregulating pathways linked to immune response, inflammation, and cytokine signalling. However, short-term cytokine treatment significantly attenuated the osteogenic and adipogenic differentiation of MSCs, as evidenced by the reduced expression of RUNX2, ALP, CEBPA, and PPARG. The priming had a negligible effect on EGF, FGF-2, HGF, LIF, and SCF secretion. The production of VEGF-A and VEGF-C was elevated, although the levels remained low. Metabolomic analysis revealed enhanced kynurenine pathway activity, indicative of increased tryptophan catabolism, accompanied by elevated levels of fatty acids and polyamines. CONCLUSIONS: Our findings demonstrate that TNF-α/IFN-γ priming reprograms WJ-MSCs by enhancing their immunomodulatory capacity at the expense of differentiation potential. These results highlight the need for tailored strategies to optimize MSC functionality for specific clinical applications.

• MeSH
• buněčná diferenciace * účinky léků   MeSH
• cytokiny * farmakologie   MeSH
• imunomodulace * účinky léků   MeSH
• interferon gama * farmakologie   MeSH
• kultivované buňky MeSH
• lidé MeSH
• mezenchymální kmenové buňky * metabolismus   cytologie   účinky léků   imunologie   MeSH
• TNF-alfa * farmakologie   MeSH
• Whartonův rosol * cytologie   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH

Electrospinning is a widely employed manufacturing platform for tissue engineering applications because it produces structures that closely mimic the extracellular matrix. Herein, we demonstrate the potential of poly(vinyl alcohol) (PVA) electrospun nanofibers as scaffolds for tissue engineering. Nanofibers were created by needleless direct current electrospinning from PVA with two different degrees of hydrolysis (DH), namely 98% and 99% and subsequently heat treated at 180 °C for up to 16 h to render them insoluble in aqueous environments without the use of toxic cross-linking agents. Despite the small differences in the PVA chemical structure, the changes in the material properties were substantial. The higher degree of hydrolysis resulted in non-woven supports with thinner fibres (285 ± 81 nm c.f. 399 ± 153 nm) that were mechanically stronger by 62% (±11%) and almost twice as more crystalline than those from 98% hydrolysed PVA. Although prolonged heat treatment (16 h) did not influence fibre morphology, it reduced the crystallinity and tensile strength for both sets of materials. All samples demonstrated a lack or very low degree of haemolysis (<5%), and there were no notable changes in their anticoagulant activity (≤3%). Thrombus formation, on the other hand, increased by 82% (±18%) for the 98% hydrolysed samples and by 71% (±10%) for the 99% hydrolysed samples, with heat treatment up to 16 h, as a direct consequence of the preservation of the fibrous morphology. 3T3 mouse fibroblasts showed the best proliferation on scaffolds that were thermally stabilised for 4 and 8 h. Overall these scaffolds show potential as 'greener' alternatives to other electrospun tissue engineering materials, especially in cases where they may be used as delivery vectors for heat tolerant additives.

• Publikační typ
• časopisecké články MeSH

The widespread use of multipotent mesenchymal stromal cell-derived secretome (MSC-sec) requires optimal preservation methods. Lyophilization offers benefits like concentrating the secretome, reducing the storage volume, and making storage conditions more flexible. This study evaluated the influence of storage duration and temperature on lyophilized MSC-sec. The conditioned medium from Wharton's jelly MSCs was stored at - 80 °C or lyophilized with or without trehalose. Lyophilized formulations were kept at - 80 °C, - 20 °C, 4 °C, or room temperature (RT) for 3 and 30 months. After storage and reconstitution, the levels of growth factors and cytokines were assessed using multiplex assay. The storage of lyophilized MSC-sec at - 80 °C ensured biomolecule preservation for 3 and 30 months. Following 3 month storage at 4 °C and RT, a notable decrease occurred in BDNF, bNGF, and sVCAM-1 levels. Prolonged 30 month storage at the same temperatures significantly reduced BDNF, bNGF, VEGF-A, IL-6, and sVCAM-1, while storage at - 20 °C decreased BDNF, bNGF, and VEGF- A levels. Trehalose supplementation of MSC-sec improved the outcome during storage at 4 °C and RT. Proper storage conditions were crucial for the preservation of lyophilized MSC-sec composition. Short-term storage at various temperatures maintained over 60% of the studied growth factors and cytokines; long-term preservation was only adequate at -80 °C.

• MeSH
• cytokiny metabolismus   MeSH
• kryoprezervace metody   MeSH
• kultivační média speciální chemie   MeSH
• kultivované buňky MeSH
• lidé MeSH
• lyofilizace * MeSH
• mezenchymální kmenové buňky * metabolismus   cytologie   MeSH
• sekretom metabolismus   MeSH
• teplota MeSH
• trehalosa metabolismus   farmakologie   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Diamond-like carbon (DLC) layers are known for their high corrosion and wear resistance, low friction, and high biocompatibility. However, it is often necessary to dope DLC layers with additional chemical elements to strengthen their adhesion to the substrate. Ti-DLC layers (doped with 0.4, 2.1, 3.7, 6.6, and 12.8 at.% of Ti) were prepared by dual pulsed laser deposition, and pure DLC, glass, and polystyrene (PS) were used as controls. In vitro cell-material interactions were investigated with an emphasis on cell adhesion, proliferation, and osteogenic differentiation. We observed slightly increasing roughness and contact angle and decreasing surface free energy on Ti-DLC layers with increasing Ti content. Three-week biological experiments were performed using adipose tissue-derived stem cells (ADSCs) and bone marrow mesenchymal stem cells (bmMSCs) in vitro. The cell proliferation activity was similar or slightly higher on the Ti-doped materials than on glass and PS. Osteogenic cell differentiation on all materials was proved by collagen and osteocalcin production, ALP activity, and Ca deposition. The bmMSCs exhibited greater initial proliferation potential and an earlier onset of osteogenic differentiation than the ADSCs. The ADSCs showed a slightly higher formation of focal adhesions, higher metabolic activity, and Ca deposition with increasing Ti content.

• MeSH
• artroplastiky kloubů * MeSH
• buněčná diferenciace MeSH
• mezenchymální kmenové buňky * metabolismus   MeSH
• osteogeneze MeSH
• povrchové vlastnosti MeSH
• titan chemie   MeSH
• uhlík chemie   MeSH
• Publikační typ
• časopisecké články MeSH

Patient-derived xenografts (PDXs) can be improved by implantation of a humanized niche. Nevertheless, the overall complexity of the current protocols, as well as the use of specific biomaterials and procedures, limits the wider adoption of this approach. Here, we identify the essential minimum steps required to create the humanized scaffolds and achieve successful acute myeloid leukemia (AML) engraftment. We compared seven biomaterials, which included both published and custom-designed materials. The highest level of bone marrow niche was achieved with extracellular matrix gels and custom collagen fiber, both of which allowed for a simple non-surgical implantation. The biomaterial selection did not influence the following AML infiltration. Regarding xenotransplantation, standard intravenous administration produced the most robust engraftment, even for two out of four otherwise non-engrafting AML samples. In contrast, direct intra-scaffold xenotransplantation did not offer any advantage. In summary, we demonstrate that the combination of an injectable biomaterial for scaffold creation plus an intravenous route for AML xenotransplantation provide the most convenient and robust approach to produce AML PDX using a humanized niche.

• MeSH
• akutní myeloidní leukemie * patologie   MeSH
• biokompatibilní materiály * farmakologie   aplikace a dávkování   MeSH
• heterografty MeSH
• lidé MeSH
• myši SCID MeSH
• myši MeSH
• tkáňové podpůrné struktury * chemie   MeSH
• transplantace heterologní * MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• myši MeSH
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH