This study investigates the impact of hydroxyapatite (HA) nanoparticles (NPs) on the cellular responses of poly(L-lactide-co-ε-caprolactone) (PLCL) scaffolds in bone tissue engineering applications. Three types of PLCL scaffolds were fabricated, varying in HANPs content. Saos-2 osteoblast-like cells (OBs) and THP-1-derived osteoclast-like cells (OCs) were co-cultured on the scaffolds, and cell proliferation was assessed using the MTS assay. The amount of double-stranded DNA (dsDNA) was quantified to evaluate cell proliferation. Expression levels of OBs and OCs markers were analyzed via quantitative polymerase chain reaction (qPCR) and the production of Collagen type I was visualized using confocal microscopy. Additionally, enzymatic activity of alkaline phosphatase (ALP) and tartrate-resistant acid phosphatase (TRAP or ACP5) was measured to assess OB and OC function, respectively. Interestingly, despite the scaffold's structured character supporting the growth of the Saos-2 OBs and THP-1-derived OCs coculture, the incorporation of HANPs did not significantly enhance cellular responses compared to scaffolds without HANPs, except for collagen type I production. These findings suggest the need for further investigation into the potential benefits of HANPs in bone tissue engineering applications. Nevertheless, our study contributes valuable insights into optimizing biomaterial design for bone tissue regeneration, with implications for drug screening and material testing protocols.

• Klíčová slova
• PLCL, bone regeneration, hydroxyapatite, osteoblasts, osteoclasts, scaffold, tissue engineering,
• MeSH
• buněčné linie MeSH
• hydroxyapatit * chemie   MeSH
• kokultivační techniky MeSH
• lidé MeSH
• nanočástice * chemie   MeSH
• nanovlákna * chemie   MeSH
• osteoblasty cytologie   metabolismus   účinky léků   MeSH
• osteogeneze MeSH
• osteoklasty cytologie   metabolismus   účinky léků   MeSH
• polyestery * chemie   MeSH
• proliferace buněk účinky léků   MeSH
• regenerace kostí * účinky léků   MeSH
• THP-1 buňky MeSH
• tkáňové inženýrství metody   MeSH
• tkáňové podpůrné struktury chemie   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• hydroxyapatit * MeSH
• poly(lactic acid-co-epsilon-caprolactone) MeSH Prohlížeč
• polyestery * MeSH

Breast cancer is the most prevalent cancer type in women worldwide. It proliferates rapidly and can metastasize into farther tissues at any stage due to the gradual invasiveness and motility of the tumor cells. These crucial properties are the outcome of the weakened intercellular adhesion, regulated by small guanosine triphosphatases (GTPases), which hydrolyze to the guanosine diphosphate (GDP)-bound conformation. We investigated the inactivating effect of ARHGAP1 on Rho GTPases involved signaling pathways after treatment with a high dose of doxorubicin. Label-free quantitative proteomic analysis of the proteome isolated from the MCF-7 breast cancer cell line, treated with 1 μM of doxorubicin, identified RAC1, CDC42, and RHOA GTPases that were inactivated by the ARHGAP1 protein. Upregulation of the GTPases involved in the transforming growth factor-beta (TGF-beta) signaling pathway initiated epithelial-mesenchymal transitions. These findings demonstrate a key role of the ARHGAP1 protein in the disruption of the cell adhesion and simultaneously allow for a better understanding of the molecular mechanism of the reduced cell adhesion leading to the subsequent metastasis. The conclusions of this study corroborate the hypothesis that chemotherapy with doxorubicin may increase the risk of metastases in drug-resistant breast cancer cells.

• Klíčová slova
• breast cancer, cell adhesion, doxorubicin, mass spectrometry, metastases, proteomics,
• MeSH
• cdc42 protein vázající GTP metabolismus   MeSH
• doxorubicin farmakologie   MeSH
• lidé MeSH
• MFC-7 buňky MeSH
• nádory prsu * farmakoterapie   MeSH
• proteiny aktivující GTPasu * metabolismus   MeSH
• proteomika MeSH
• rac1 protein vázající GTP metabolismus   MeSH
• Rho proteiny vázající GTP * metabolismus   MeSH
• rhoA protein vázající GTP metabolismus   MeSH
• Check Tag
• lidé MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• ARHGAP1 protein, human MeSH Prohlížeč
• cdc42 protein vázající GTP MeSH
• doxorubicin MeSH
• proteiny aktivující GTPasu * MeSH
• rac1 protein vázající GTP MeSH
• Rho proteiny vázající GTP * MeSH
• rhoA protein vázající GTP MeSH

Treatment of complete loss of skin thickness requires expensive cellular materials and limited skin grafts used as temporary coverage. This paper presents an acellular bilayer scaffold modified with polydopamine (PDA), which is designed to mimic a missing dermis and a basement membrane (BM). The alternate dermis is made from freeze-dried collagen and chitosan (Coll/Chit) or collagen and a calcium salt of oxidized cellulose (Coll/CaOC). Alternate BM is made from electrospun gelatin (Gel), polycaprolactone (PCL), and CaOC. Morphological and mechanical analyzes have shown that PDA significantly improved the elasticity and strength of collagen microfibrils, which favorably affected swelling capacity and porosity. PDA significantly supported and maintained metabolic activity, proliferation, and viability of the murine fibroblast cell lines. The in vivo experiment carried out in a domestic Large white pig model resulted in the expression of pro-inflammatory cytokines in the first 1-2 weeks, giving the idea that PDA and/or CaOC trigger the early stages of inflammation. Otherwise, in later stages, PDA caused a reduction in inflammation with the expression of the anti-inflammatory molecule IL10 and the transforming growth factor β (TGFβ1), which could support the formation of fibroblasts. Similarities in treatment with native porcine skin suggested that the bilayer can be used as an implant for full-thickness skin wounds and thus eliminate the use of skin grafts.

• Klíčová slova
• Bilayer, Chitosan, Collagen, Oxidized cellulose, Polydopamine, Wound healing,
• MeSH
• myši MeSH
• nanovlákna * MeSH
• prasata MeSH
• sloučeniny osmia MeSH
• zánět MeSH
• zvířata MeSH
• Check Tag
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• chloropentaammineosmium(III) chloride MeSH Prohlížeč
• polydopamine MeSH Prohlížeč
• sloučeniny osmia MeSH

PURPOSE: Osteoporosis is a severe health problem with social and economic impacts on society. The standard treatment consists of the systemic administration of drugs such as bisphosphonates, with alendronate (ALN) being one of the most common. Nevertheless, complications of systemic administration occur with this drug. Therefore, it is necessary to develop new strategies, such as local administration. METHODS: In this study, emulsion/dispersion scaffolds based on W/O emulsion of PCL and PF68 with ALN, containing hydroxyapatite (HA) nanoparticles as the dispersion phase were prepared using electrospinning. Scaffolds with different release kinetics were tested in vitro on the co-cultures of osteoblasts and osteoclast-like cells, isolated from adult osteoporotic and control rats. Cell viability, proliferation, ALP, TRAP and CA II activity were examined. A scaffold with a gradual release of ALN was tested in vivo in the bone defects of osteoporotic and control rats. RESULTS: The release kinetics were dependent on the scaffold composition and the used system of the poloxamers. The ALN was released from the scaffolds for more than 22 days. The behavior of cells cultured in vitro on scaffolds with different release kinetics was comparable. The difference was evident between cell co-cultures isolated from osteoporotic and control animals. The PCL/HA scaffold show slow degradation in vivo and residual scaffold limited new bone formation inside the defects. Nevertheless, the released ALN supported bone formation in the areas surrounding the residual scaffold. Interestingly, a positive effect of systemic administration of ALN was not proved. CONCLUSION: The prepared scaffolds enabled tunable control release of ALN. The effect of ALN was proved in vitro and in in vivo study supported peri-implant bone formation.

• Klíčová slova
• alendronate, co-culture, drug delivery system, fibrous scaffold, osteoporosis,
• MeSH
• alendronát * farmakologie   MeSH
• emulze farmakologie   MeSH
• hydroxyapatit farmakologie   MeSH
• inhibitory kostní resorpce * farmakologie   MeSH
• krysa rodu Rattus MeSH
• osteoblasty MeSH
• osteogeneze MeSH
• osteoklasty MeSH
• zvířata MeSH
• Check Tag
• krysa rodu Rattus MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• alendronát * MeSH
• emulze MeSH
• hydroxyapatit MeSH
• inhibitory kostní resorpce * MeSH

Titanium surface treated with titanium oxide nanotubes was used in many studies to quantify the effect of surface topography on cell fate. However, the predicted optimal diameter of nanotubes considerably differs among studies. We propose a model that explains cell adhesion to a nanostructured surface by considering the deformation energy of cell protrusions into titanium nanotubes and the adhesion to the surface. The optimal surface topology is defined as a geometry that gives the membrane a minimum energy shape. A dimensionless parameter, the cell interaction index, was proposed to describe the interplay between the cell membrane bending, the intrinsic curvature, and the strength of cell adhesion. Model simulation shows that an optimal nanotube diameter ranging from 20 nm to 100 nm (cell interaction index between 0.2 and 1, respectively) is feasible within a certain range of parameters describing cell membrane adhesion and bending. The results indicate a possibility to tune the topology of a nanostructural surface in order to enhance the proliferation and differentiation of cells mechanically compatible with the given surface geometry while suppressing the growth of other mechanically incompatible cells.

• Klíčová slova
• Titanium, adhesion, bending, biomechanics, cell membrane, nanotubes, surface energy,
• MeSH
• buněčná adheze MeSH
• buněčná membrána MeSH
• nanotrubičky * chemie   MeSH
• povrchové vlastnosti MeSH
• proliferace buněk MeSH
• titan * farmakologie   chemie   MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• titan * MeSH
• titanium dioxide MeSH Prohlížeč

Chronic wounds represent a significant socio-economic problem, and the improvement of their healing is therefore an essential issue. This paper describes the preparation and biological properties of a novel functionalized nanofiber wound dressing consisting of a polycaprolactone nanofiber carrier modified by a drug delivery system, based on the lipid particles formed by 1-tetradecanol and encapsulated gentamicin and tocopherol acetate. The cytotoxicity of extracts was tested using a metabolic activity assay, and the antibacterial properties of the extracts were tested in vitro on the bacterial strains Staphylococcus aureus and Pseudomonas aeruginosa. The effect of the wound dressing on chronic wound healing was subsequently tested using a mouse model. Fourteen days after surgery, the groups treated by the examined wound cover showed a lower granulation, reepithelization, and inflammation score compared to both the uninfected groups, a lower dermis organization compared to the control, a higher scar thickness compared to the other groups, and a higher thickness of hypodermis and bacteria score compared to both the uninfected groups. This work demonstrates the basic parameters of the safety (biocompatibility) and performance (effect on healing) of the dressing as a medical device and indicates the feasibility of the concept of its preparation in outpatient conditions using a suitable functionalization device.

• Klíčová slova
• antibacterial, antioxidant, encapsulation, in vivo, nanofiber substrate, personalized care, wound dressing,
• Publikační typ
• časopisecké články MeSH

Co-cultures of osteoblasts and osteoclasts are on the rise because they enable a more complex study. Diseases such as osteoporosis are related to a higher age. Thus, cell isolation from adult individuals is necessary. Osteoblasts can be isolated from the rat femur by three methods: explant culture, explant culture with enzymatic pre-treatment, or enzymatic treatment. The isolation methods yield different populations of osteoblasts which, in a co-culture with peripheral blood mononuclear cells, might result in differences in osteoclastogenesis. Therefore, we examined the differences in osteogenic markers, cell proliferation, and the metabolic activity of isolated osteoblast-like cells in a growth and differentiation medium. We then evaluated the effect of the isolated populations of osteoblast-like cells on osteoclastogenesis in a subsequent co-culture by evaluating osteoclast markers, counting formed osteoclast-like cells, and analyzing their area and number of nuclei. Co-cultures were performed in the presence or absence of osteoclastogenic growth factors, M-CSF and RANKL. It was discovered that enzymatic isolation is not feasible in adult rats, but explant culture and explant culture with enzymatic pre-treatment were both successful. Explant culture with enzymatic pre-treatment yielded cells with a higher proliferation than explant culture in a growth medium. The differentiation medium reduced differences in proliferation during the culture. Some differences in metabolic activity and ALP activity were also found between the osteoblast-like cells isolated by explant culture or by explant culture with enzymatic pre-treatment, but only on some days of cultivation. According to microscopy, the presence of exogenous growth factors supporting osteoclastogenesis in co-cultures was necessary for the formation of osteoclast-like cells. In this case, the formation of a higher number of osteoclast-like cells with a larger area was observed in the co-culture with osteoblast-like cells isolated by explant culture compared to the explant culture with enzymatic pre-treatment. Apart from this observation, no differences in osteoclast markers were noted between the co-cultures with osteoblast-like cells isolated by explant culture and the explant culture with enzymatic pre-treatment. The TRAP and CA II activity was higher in the co-cultures with exogenous growth than that in the co-cultures without exogenous growth factors on day 7, but the opposite was true on day 14. To conclude, explant culture and explant culture with enzymatic pre-treatment are both suitable methods to yield osteoblast-like cells from adult rats capable of promoting osteoclastogenesis in a direct co-culture with peripheral blood mononuclear cells. Explant culture with enzymatic pre-treatment yielded cells with a higher proliferation. The explant culture yielded osteoblast-like cells which induced the formation of a higher number of osteoclast-like cells with a larger area compared to the explant culture with enzymatic pre-treatment when cultured with exogenous M-CSF and RANKL.

• Klíčová slova
• adult rat, co-culture, mature bone, osteoblast-like cell, osteoclast-like cell,
• MeSH
• buněčná diferenciace MeSH
• faktor stimulující kolonie makrofágů * metabolismus   MeSH
• kokultivační techniky MeSH
• krysa rodu Rattus MeSH
• kultivované buňky MeSH
• leukocyty mononukleární metabolismus   MeSH
• ligand RANK metabolismus   MeSH
• osteoblasty metabolismus   MeSH
• osteogeneze * MeSH
• osteoklasty metabolismus   MeSH
• zvířata MeSH
• Check Tag
• krysa rodu Rattus MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• faktor stimulující kolonie makrofágů * MeSH
• ligand RANK MeSH

Electrospun hybrid nanofibers, based on functional agents immobilized in polymeric matrix, possess a unique combination of collective properties. These are beneficial for a wide range of applications, which include theranostics, filtration, catalysis, and tissue engineering, among others. The combination of functional agents in a nanofiber matrix offer accessibility to multifunctional nanocompartments with significantly improved mechanical, electrical, and chemical properties, along with better biocompatibility and biodegradability. This review summarizes recent work performed for the fabrication, characterization, and optimization of different hybrid nanofibers containing varieties of functional agents, such as laser ablated inorganic nanoparticles (NPs), which include, for instance, gold nanoparticles (Au NPs) and titanium nitride nanoparticles (TiNPs), perovskites, drugs, growth factors, and smart, inorganic polymers. Biocompatible and biodegradable polymers such as chitosan, cellulose, and polycaprolactone are very promising macromolecules as a nanofiber matrix for immobilizing such functional agents. The assimilation of such polymeric matrices with functional agents that possess wide varieties of characteristics require a modified approach towards electrospinning techniques such as coelectrospinning and template spinning. Additional focus within this review is devoted to the state of the art for the implementations of these approaches as viable options for the achievement of multifunctional hybrid nanofibers. Finally, recent advances and challenges, in particular, mass fabrication and prospects of hybrid nanofibers for tissue engineering and biomedical applications have been summarized.

• Klíčová slova
• bone regeneration, drug delivery, electrospinning, functional agents, hybrid nanofibers, nanomedicine, nanoparticles, tissue engineering,
• Publikační typ
• časopisecké články MeSH
• přehledy 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

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