Several in vitro models have been developed to mimic chronic lymphocytic leukemia (CLL) proliferation in immune niches; however, they typically do not induce robust proliferation. We prepared a novel model based on mimicking T-cell signals in vitro and in patient-derived xenografts (PDXs). Six supportive cell lines were prepared by engineering HS5 stromal cells with stable expression of human CD40L, IL4, IL21, and their combinations. Co-culture with HS5 expressing CD40L and IL4 in combination led to mild CLL cell proliferation (median 7% at day 7), while the HS5 expressing CD40L, IL4, and IL21 led to unprecedented proliferation rate (median 44%). The co-cultures mimicked the gene expression fingerprint of lymph node CLL cells (MYC, NFκB, and E2F signatures) and revealed novel vulnerabilities in CLL-T-cell-induced proliferation. Drug testing in co-cultures revealed for the first time that pan-RAF inhibitors fully block CLL proliferation. The co-culture model can be downscaled to five microliter volume for large drug screening purposes or upscaled to CLL PDXs by HS5-CD40L-IL4 ± IL21 co-transplantation. Co-transplanting NSG mice with purified CLL cells and HS5-CD40L-IL4 or HS5-CD40L-IL4-IL21 cells on collagen-based scaffold led to 47% or 82% engraftment efficacy, respectively, with ~20% of PDXs being clonally related to CLL, potentially overcoming the need to co-transplant autologous T-cells in PDXs.

• MeSH
• buňky stromatu * metabolismus   patologie   MeSH
• chronická lymfatická leukemie * patologie   genetika   farmakoterapie   MeSH
• inhibitory proteinkinas farmakologie   MeSH
• interleukiny genetika   metabolismus   MeSH
• kokultivační techniky * MeSH
• lidé MeSH
• ligand CD40 * metabolismus   genetika   MeSH
• myši MeSH
• proliferace buněk * MeSH
• T-lymfocyty imunologie   metabolismus   MeSH
• xenogenní modely - testy antitumorózní aktivity MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• inhibitory proteinkinas MeSH
• Interleukin-21 MeSH
• interleukiny MeSH
• ligand CD40 * MeSH

Collagen I-based foams were modified with calcined or noncalcined hydroxyapatite or calcium phosphates with various particle sizes and pores to monitor their effect on cell interactions. The resulting scaffolds thus differed in grain size, changing from nanoscale to microscopic, and possessed diverse morphological characteristics and resorbability. The materials' biological action was shown on human bone marrow MSCs. Scaffold morphology was identified by SEM. Using viability test, qPCR, and immunohistochemical staining, we evaluated the biological activity of all of the materials. This study revealed that the most suitable scaffold composition for osteogenesis induction is collagen I foam with calcined hydroxyapatite with a pore size of 360 ± 130 µm and mean particle size of 0.130 µm. The expression of osteogenic markers RunX2 and ColI mRNA was promoted, and a strong synthesis of extracellular protein osteocalcin was observed. ColI/calcined HAP scaffold showed significant osteogenic potential, and can be easily manipulated and tailored to the defect size, which gives it great potential for bone tissue engineering applications.

• Klíčová slova
• bioceramics, collagen, osteogenesis,
• MeSH
• buněčná diferenciace MeSH
• hydroxyapatit * chemie   farmakologie   MeSH
• kolagen typu I genetika   MeSH
• kultivované buňky MeSH
• lidé MeSH
• osteogeneze * MeSH
• tkáňové inženýrství metody   MeSH
• tkáňové podpůrné struktury chemie   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• hydroxyapatit * MeSH
• kolagen typu I MeSH

Many growth factors have been studied as additives accelerating lumbar fusion rates in different animal models. However, their low hydrolytic and thermal stability both in vitro and in vivo limits their workability and use. In the proposed work, a stabilized vasculogenic and prohealing fibroblast growth factor-2 (FGF2-STAB®) exhibiting a functional half-life in vitro at 37 °C more than 20 days was applied for lumbar fusion in combination with a bioresorbable scaffold on porcine models. An experimental animal study was designed to investigate the intervertebral fusion efficiency and safety of a bioresorbable ceramic/biopolymer hybrid implant enriched with FGF2-STAB® in comparison with a tricortical bone autograft used as a gold standard. Twenty-four experimental pigs underwent L2/3 discectomy with implantation of either the tricortical iliac crest bone autograft or the bioresorbable hybrid implant (BHI) followed by lateral intervertebral fixation. The quality of spinal fusion was assessed by micro-computed tomography (micro-CT), biomechanical testing, and histological examination at both 8 and 16 weeks after the surgery. While 8 weeks after implantation, micro-CT analysis demonstrated similar fusion quality in both groups, in contrast, spines with BHI involving inorganic hydroxyapatite and tricalcium phosphate along with organic collagen, oxidized cellulose, and FGF2- STAB® showed a significant increase in a fusion quality in comparison to the autograft group 16 weeks post-surgery (p = 0.023). Biomechanical testing revealed significantly higher stiffness of spines treated with the bioresorbable hybrid implant group compared to the autograft group (p < 0.05). Whilst histomorphological evaluation showed significant progression of new bone formation in the BHI group besides non-union and fibrocartilage tissue formed in the autograft group. Significant osteoinductive effects of BHI based on bioceramics, collagen, oxidized cellulose, and FGF2-STAB® could improve outcomes in spinal fusion surgery and bone tissue regeneration.

• Klíčová slova
• FGF2, animal model, autograft, biomechanics, ceramic, collagen, histology, lumbar spinal fusion, micro-CT, tissue engineering,
• Publikační typ
• časopisecké články MeSH

Wound healing is a process regulated by a complex interaction of multiple growth factors including fibroblast growth factor 2 (FGF2). Although FGF2 appears in several tissue engineered studies, its applications are limited due to its low stability both in vitro and in vivo. Here, this shortcoming is overcome by a unique nine-point mutant of the low molecular weight isoform FGF2 retaining full biological activity even after twenty days at 37 °C. Crosslinked freeze-dried 3D porous collagen/chitosan scaffolds enriched with this hyper stable recombinant human protein named FGF2-STAB® were tested for in vitro biocompatibility and cytotoxicity using murine 3T3-A31 fibroblasts, for angiogenic potential using an ex ovo chick chorioallantoic membrane assay and for wound healing in vivo with 3-month old white New Zealand rabbits. Metabolic activity assays indicated the positive effect of FGF2-STAB® already at very low concentrations (0.01 µg/mL). The angiogenic properties examined ex ovo showed enhanced vascularization of the tested scaffolds. Histological evaluation and gene expression analysis by RT-qPCR proved newly formed granulation tissue at the place of a previous skin defect without significant inflammation infiltration in vivo. This work highlights the safety and biocompatibility of newly developed crosslinked collagen/chitosan scaffolds involving FGF2-STAB® protein. Moreover, these sponges could be used as scaffolds for growing cells for dermis replacement, where neovascularization is a crucial parameter for successful skin regeneration.

• Klíčová slova
• FGF2, chitosan, collagen, scaffold, skin regeneration, tissue engineering,
• Publikační typ
• časopisecké články MeSH

A highly porous scaffold is a desirable outcome in the field of tissue engineering. The porous structure mediates water-retaining properties that ensure good nutrient transportation as well as creates a suitable environment for cells. In this study, porous antibacterial collagenous scaffolds containing chitosan and selenium nanoparticles (SeNPs) as antibacterial agents were studied. The addition of antibacterial agents increased the application potential of the material for infected and chronic wounds. The morphology, swelling, biodegradation, and antibacterial activity of collagen-based scaffolds were characterized systematically to investigate the overall impact of the antibacterial additives. The additives visibly influenced the morphology, water‑retaining properties as well as the stability of the materials in the presence of collagenase enzymes. Even at concentrations as low as 5 ppm of SeNPs, modified polymeric scaffolds showed considerable inhibition activity towards Gram-positive bacterial strains such as Staphylococcus aureus and methicillin-resistant Staphylococcus aureus and Staphylococcus epidermidis in a dose-dependent manner.

• Klíčová slova
• Staphylococcus, bacteria, chitosan, collagen, drug release, freeze-drying, infected burn injuries, selenium nanoparticles, tissue engineering,
• Publikační typ
• časopisecké články MeSH

Titanium and its alloys are widely used for substitution of hard tissues, especially in orthopaedic and dental surgery. Despite the benefit of the use of titanium for such applications, there are still questions which must be sorted out. Surface properties are crucial for cell adhesion, proliferation and differentiation. Mainly, micro/nanostructured surfaces positively influence osteogenic differentiation of human mesenchymal stem cells. Ti6Al4V is a biocompatible α + β alloy which is widely used in orthopaedics. The aim of this study was to investigate the interaction of the nanostructured and ground Ti6Al4V titanium alloys with simulated body fluid complemented by the defined precipitation of hydroxyapatite-like coating and to study the cytotoxicity and differentiation capacity of cells with such a modified titanium alloy. Nanostructures were fabricated using electrochemical oxidation. Human mesenchymal stem cells (hMSC) were used to evaluate cell adhesion, metabolic activity and proliferation on the specimens. The differentiation potential of the samples was investigated using PCR and specific staining of osteogenic markers collagen type I and osteocalcin. Our results demonstrate that both pure Ti6Al4V, nanostructured samples, and hydroxyapatite-like coating supported hMSC growth and metabolic activity. Nanostructured samples improved collagen type I synthesis after 14 days, while both nanostructured and hydroxyapatite-like coated samples enhanced collagen synthesis on day 21. Osteocalcin synthesis was the most enhanced by hydroxyapatite-like coating on the nanostructured surfaces. Our results indicate that hydroxyapatite-like coating is a useful tool guiding hMSC osteogenic differentiation.

• Publikační typ
• časopisecké články MeSH

In this work we have used X-ray micro-computed tomography (μCT) as a method to observe the morphology of 3D porous pure collagen and collagen-composite scaffolds useful in tissue engineering. Two aspects of visualizations were taken into consideration: improvement of the scan and investigation of its sensitivity to the scan parameters. Due to the low material density some parts of collagen scaffolds are invisible in a μCT scan. Therefore, here we present different contrast agents, which increase the contrast of the scanned biopolymeric sample for μCT visualization. The increase of contrast of collagenous scaffolds was performed with ceramic hydroxyapatite microparticles (HAp), silver ions (Ag(+)) and silver nanoparticles (Ag-NPs). Since a relatively small change in imaging parameters (e.g. in 3D volume rendering, threshold value and μCT acquisition conditions) leads to a completely different visualized pattern, we have optimized these parameters to obtain the most realistic picture for visual and qualitative evaluation of the biopolymeric scaffold. Moreover, scaffold images were stereoscopically visualized in order to better see the 3D biopolymer composite scaffold morphology. However, the optimized visualization has some discontinuities in zoomed view, which can be problematic for further analysis of interconnected pores by commonly used numerical methods. Therefore, we applied the locally adaptive method to solve discontinuities issue. The combination of contrast agent and imaging techniques presented in this paper help us to better understand the structure and morphology of the biopolymeric scaffold that is crucial in the design of new biomaterials useful in tissue engineering.

• MeSH
• biokompatibilní materiály chemie   MeSH
• hydroxyapatit chemie   MeSH
• kolagen chemie   MeSH
• kontrastní látky MeSH
• kovové nanočástice chemie   MeSH
• rentgenová mikrotomografie * MeSH
• stříbro chemie   MeSH
• tkáňové podpůrné struktury chemie   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• biokompatibilní materiály MeSH
• hydroxyapatit MeSH
• kolagen MeSH
• kontrastní látky MeSH
• stříbro MeSH