Bone tissue engineering tries to simulate natural behavior of hard tissues. This study aimed to produce scaffolds based on polyvinyl alcohol (PVA) and hyaluronic acid (HA) with hydroxyapatite (HAp) incorporated in two different ways, by in situ synthesis and physical mixing of pre-prepared HAp. In situ synthesis resulted in calcium deficient form of HAp with lower crystallinity. The proliferation of human osteoblast-like cells MG-63 proved to be better in the scaffolds with in situ synthesized HAp compared to those with physically mixed pre-prepared HAp. For scaffolds with PVA/HA/HAp ratio 3:1:2, there was significantly higher initial adhesion (p = 0.0440), as well as the proliferation in the following days (p < 0.001). It seemed to be advantageous improve the properties of the scaffold by in situ synthesizing of HAp directly in the organic matrix.

• MeSH
• biokompatibilní materiály chemie   MeSH
• buněčná adheze MeSH
• hemolýza MeSH
• hydrogely chemie   MeSH
• hydroxyapatit chemie   MeSH
• kyselina hyaluronová chemie   MeSH
• lidé MeSH
• osteoblasty cytologie   MeSH
• polyvinylalkohol chemie   MeSH
• proliferace buněk MeSH
• testování materiálů MeSH
• tkáňové inženýrství MeSH
• tkáňové podpůrné struktury chemie   MeSH
• viabilita buněk MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH

The protein heterogeneity at the single-cell level has been recognized to be vital for an understanding of various life processes during animal development. In addition, the knowledge of accurate quantity of relevant proteins at cellular level is essential for appropriate interpretation of diagnostic and therapeutic results. Some low-copy-number proteins are known to play a crucial role during cell proliferation, differentiation, and also in apoptosis. The fate decision is often based on the concentration of these proteins in the individual cells. This is likely to apply also for caspases, cysteine proteases traditionally associated with cell death via apoptosis but recently being discovered also as important factors in cell proliferation and differentiation. The hypothesis was tested in bone-related cells, where modulation of fate from apoptosis to proliferation/differentiation and vice versa is particularly challenging, e.g., towards anti-osteoporotic treatments and anti-cancer strategies. An ultrasensitive and highly selective method based on bioluminescence photon counting was used to quantify activated caspase-3/7 in order to demonstrate protein-level heterogeneity in individual cells within one population and to associate quantitative measurements with different cell fates (proliferation, differentiation, apoptosis). The results indicate a gradual increase of caspase-3/7 activation from the proliferative status to differentiation (more than three times) and towards apoptosis (more than six times). The findings clearly support one of the putative key mechanisms of non-apoptotic functions of pro-apoptotic caspases based on fine-tuning of their activation levels.

• MeSH
• aktivace enzymů MeSH
• apoptóza MeSH
• buněčná diferenciace MeSH
• buněčné linie MeSH
• kaspasa 3 chemie   genetika   metabolismus   MeSH
• kaspasa 7 chemie   genetika   metabolismus   MeSH
• myši MeSH
• osteoblasty cytologie   fyziologie   MeSH
• proliferace buněk MeSH
• zvířata MeSH
• Check Tag
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH

Caspases are proteases traditionally associated with inflammation and cell death. Recently, they have also been shown to modulate cell proliferation and differentiation. The aim of the current research was to search for osteogenic molecules affected by caspase inhibition and to specify the individual caspases critical for these effects with a focus on proapoptotic caspases: caspase-2, -3, -6, -7, -8 and -9. Along with osteocalcin (Ocn), general caspase inhibition significantly decreased the expression of the Phex gene in differentiated MC3T3-E1 cells. The inhibition of individual caspases indicated that caspase-8 is a major contributor to the modification of Ocn and Phex expression. Caspase-2 and-6 had effects on Ocn and caspase-6 had an effect on Phex. These data confirm and expand the current knowledge about the nonapoptotic roles of caspases and the effect of their pharmacological inhibition on the osteogenic potential of osteoblastic cells.

• MeSH
• buněčné linie MeSH
• inhibitory kaspas farmakologie   MeSH
• kaspasy metabolismus   MeSH
• myši MeSH
• neutrální endopeptidasa regulující fosfáty metabolismus   MeSH
• osteoblasty cytologie   metabolismus   MeSH
• osteogeneze účinky léků   MeSH
• osteokalcin metabolismus   MeSH
• zvířata MeSH
• Check Tag
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Sprouty proteins are modulators of the MAPK/ERK pathway. Amongst these, Sprouty2 (SPRY2) has been investigated as a possible factor that takes part in the initial phases of osteogenesis. However, the in vivo context has not yet been investigated and the underlying mechanisms taking place in vitro remain unknown. Therefore, in this study, the impact of Spry2 deficiency was examined in the developing tibias of Spry2 deficient (-/-) mouse. The investigation was performed when the osteogenic zone became clearly visible and when all three basic bone cells types were present. The main markers of osteoblasts, osteocytes and osteoclasts were evaluated by immunohistochemistry and RT-PCR. RT-PCR showed that the expression of Sost was 3.5 times higher in Spry2-/- than in the wild-type bone, which pointed to a still unknown mechanism of action of SPRY2 on the differentiation of osteocytes. The up-regulation of Sost was independent of Hif-1α expression and could not be related to its positive regulator, Runx2, since none of these factors showed an increased expression in the bone of Spry2-/- mice. Regarding the RANK/RANKL/OPG pathway, the Spry2-/- showed an increased expression of Rank, but no significant change in the expression of Rankl and Opg. Thanks to these results, the impact of Spry2 deletion is shown for the first time in the developing bone as a complex organ including, particularly, an effect on osteoblasts (Runx2) and osteocytes (Sost). This might explain the previously reported decrease in bone formation in postnatal Spry2-/- mice.

• MeSH
• buněčná diferenciace MeSH
• cytoplazma metabolismus   MeSH
• faktor 1 indukovatelný hypoxií - podjednotka alfa metabolismus   MeSH
• ligand RANK metabolismus   MeSH
• membránové proteiny genetika   fyziologie   MeSH
• myši inbrední ICR MeSH
• myši knockoutované MeSH
• myši MeSH
• osteoblasty cytologie   metabolismus   MeSH
• osteocyty cytologie   metabolismus   MeSH
• osteogeneze * MeSH
• osteoklasty cytologie   metabolismus   MeSH
• osteoprotegerin metabolismus   MeSH
• proliferace buněk MeSH
• protein-serin-threoninkinasy genetika   fyziologie   MeSH
• vývoj kostí MeSH
• vývojová regulace genové exprese * MeSH
• zvířata MeSH
• Check Tag
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

The present work focuses on the development of novel multicomponent organic-inorganic hydrogel composites for bone tissue engineering. For the first time, combination of the organic components commonly used in food industry, namely whey protein isolate (WPI) and gelatin from bovine skin, as well as inorganic material commonly used as a major component of hydraulic bone cements, namely α-TCP in various concentrations (0-70 wt%) was proposed. The results showed that α-TCP underwent incomplete transformation to calcium-deficient hydroxyapatite (CDHA) during preparation process of the hydrogels. Microcomputer tomography showed inhomogeneous distribution of the calcium phosphate (CaP) phase in the resulting composites. Nevertheless, hydrogels containing 30-70 wt% α-TCP showed significantly improved mechanical properties. The values of Young's modulus and the stresses corresponding to compression of a sample by 50% increased almost linearly with increasing concentration of ceramic phase. Incomplete transformation of α-TCP to CDHA during preparation process of composites provides them high reactivity in simulated body fluid during 14-day incubation. Preliminary in vitro studies revealed that the WPI/gelatin/CaP composite hydrogels support the adhesion, spreading, and proliferation of human osteoblast-like MG-63 cells. The WPI/gelatin/CaP composite hydrogels obtained in this work showed great potential for the use in bone tissue engineering and regenerative medicine applications.

• MeSH
• buněčné linie MeSH
• fosforečnany vápenaté * chemie   farmakologie   MeSH
• hydrogely * chemie   farmakologie   MeSH
• kosti a kostní tkáň cytologie   metabolismus   MeSH
• lidé MeSH
• osteoblasty cytologie   metabolismus   MeSH
• syrovátkové proteiny * chemie   farmakologie   MeSH
• tkáňové inženýrství * MeSH
• želatina * chemie   farmakologie   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Hafnium dioxide (HfO2) is attracting attention for bio-related applications due to its good cytocompatibility, high density, and resistance to corrosion and mechanical damage. Here we synthesize two types of hafnium-oxide thin films on substrates via self-organized electrochemical anodization: (1) an array of hierarchically structured nanorods anchored to a thin oxide layer and (2) a microscopically flat oxide film. The nanostructured film is composed of a unique mixture of HfO2, suboxide Hf2O3, and oxide-hydroxide compound HfO2·nH2O whereas the flat film is mainly HfO2. In vitro interaction of the two films with MG-63 osteoblast-like cells and Gram-negative E. coli bacteria is studied for the first time to assess the potential of the films for biomedical application. Both films reveal good cytocompatibility and affinity for proteins, represented by fibronectin and especially albumin, which is absorbed in a nine times larger amount. The morphology and specific surface chemistry of the nanostructured film cause a two-fold enhanced antibacterial effect, better cell attachment, significantly improved proliferation of cells, five-fold rise in the cellular Young's modulus, slightly stronger production of reactive oxygen species, and formation of cell clusters. Compared with the flat film, the nanostructured one features the weakening of AFM-measured adhesion force at the cell/surface interface, probably caused by partially lifting the nanorods from the substrate due to the strong contact with cells. The present findings deepen the understanding of biological processes at the living cell/metal-oxide interface, underlying the role of surface chemistry and the impact of nanostructuring at the nanoscale.

• MeSH
• biokompatibilní materiály farmakologie   MeSH
• buněčné linie MeSH
• Escherichia coli účinky léků   MeSH
• hafnium * chemie   farmakologie   MeSH
• lidé MeSH
• nanostruktury chemie   terapeutické užití   MeSH
• osteoblasty cytologie   účinky léků   MeSH
• oxidy * chemie   farmakologie   MeSH
• povrchové vlastnosti MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

A combination of hard sphere and high internal phase emulsion templating gives a platform for synthesizing hierarchically porous polymers with a unique topology exhibiting interconnected spherical features on multiple levels. Polymeric spheres are fused by thermal sintering to create a 3D monolithic structure while an emulsion with a high proportion of internal phase and monomers in the continuous phase is added to the voids of the previously constructed monolith. Following polymerization of the emulsion and dissolution of the templating structure, a down-replicating topology is created with a primary level of pores as a result of fused spheres of the 3D monolithic structure, a secondary level of pores resulting from the emulsion's internal phase, and a tertiary level of interconnecting channels. Thiol-ene chemistry with divinyladipate and pentaerythritol tetrakis(3-mercaptopropionate) is used to demonstrate the preparation of a crosslinked polyester with overall porosity close to 90%. Due to multilevel porosity, such materials are interesting for applications in bone tissue engineering, possibly simulating the native sponge like bone structure. Their potential to promote ossteointegration is tested using human bone derived osteoblasts. Material-cell interactions are evaluated and they reveal growth and proliferation of osteoblasts both on surface and in the bulk of the scaffold.

• MeSH
• emulze chemie   MeSH
• kosti a kostní tkáň fyziologie   MeSH
• lidé MeSH
• modul pružnosti MeSH
• osteoblasty cytologie   MeSH
• polymethylmethakrylát chemie   MeSH
• poréznost MeSH
• teplota MeSH
• tkáňové inženýrství metody   MeSH
• tkáňové podpůrné struktury chemie   MeSH
• tvrdost MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

The application of biodegradable magnesium-based materials in the biomedical field is highly restricted by their low fatigue strength and high corrosion rate in biological environments. Herein, we treated the surface of a biocompatible magnesium alloy AZ31 by severe shot peening in order to evaluate the potential of surface grain refinement to enhance this alloy's functionality in a biological environment. The AZ31 samples were studied in terms of micro/nanostructural, mechanical, and chemical characteristics in addition to cytocompatibility properties. The evolution of surface grain structure and surface morphology were investigated using optical, scanning and transmission electron microscopy. Surface roughness, wettability, and chemical composition, as well as in depth-microhardness and residual stress distribution, fatigue behaviour and corrosion resistance were investigated. Cytocompatibility tests with osteoblasts (bone forming cells) were performed using sample extracts. The results revealed for the first time that severe shot peening can significantly enhance mechanical properties of AZ31 without causing adverse effects on the growth of surrounding osteoblasts. The corrosion behavior, on the other hand, was not improved; nevertheless, removing the rough surface layer with a high density of crystallographic lattice defects, without removing the entire nanocrystallized layer, provided a good potential for improving corrosion characteristics after severe shot peening and thus, this method should be studied for a wide range of orthopedic applications in which biodegradable magnesium is used. STATEMENT OF SIGNIFICANCE: A major challenge for most commonly used metals for bio-implants is their non-biodegradability that necessitates revision surgery for implant retrieval when used as fixation plates, screws, etc. Magnesium is reported among the most biocompatible metals that resorb over time without adverse tissue reactions and is indispensable for many biochemical processes in human body. However, fast and uncontrolled degradation of magnesium alloys in the physiological environment in addition to their inadequate mechanical properties especially under repeated loading have limited their application in the biomedical field. The present study providesdata on the effect of a relatively simple surface nanocrystallziation method with high potential to tailor the mechanical and chemical behavior of magnesium based material while maintaining its cytocompatibility.

• MeSH
• elektrochemie MeSH
• koroze MeSH
• kultivované buňky MeSH
• lidé MeSH
• nanostruktury chemie   ultrastruktura   MeSH
• osteoblasty cytologie   účinky léků   MeSH
• povrchové vlastnosti MeSH
• slitiny farmakologie   MeSH
• testování materiálů metody   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Recently, milk-derived proteins have attracted attention for applications in the biomedical field such as tissue regeneration. Whey protein isolate (WPI), especially its main component β-lactoglobulin, can modulate immunity and acts as an antioxidant, antitumor, antiviral, and antibacterial agent. There are very few reports of the application of WPI in tissue engineering, especially in bone tissue engineering. In this study, we tested the influence of different concentrations of WPI on behavior of human osteoblast-like Saos-2 cells, human adipose tissue-derived stem cells (ASC), and human neonatal dermal fibroblasts (FIB). The positive effect on growth was apparent for Saos-2 cells and FIB but not for ASC. However, the expression of markers characteristic for early osteogenic cell differentiation [type-I collagen (COL1) and alkaline phosphatase (ALP)] as well as ALP activity, increased dose-dependently in ASC. Importantly, Saos-2 cells were able to deposit calcium in the presence of WPI, even in a proliferation medium without other supplements that support osteogenic cell differentiation. The results indicate that, depending on the cell type, WPI can act as an enhancer of cell proliferation and osteogenic differentiation. Therefore, enrichment of biomaterials for bone regeneration with WPI seems a promising approach, especially due to the low cost of WPI.

• MeSH
• alkalická fosfatasa metabolismus   MeSH
• buněčná diferenciace MeSH
• kmenové buňky cytologie   metabolismus   MeSH
• kolagen typu I metabolismus   MeSH
• kultivované buňky MeSH
• lidé MeSH
• osteoblasty cytologie   metabolismus   MeSH
• osteogeneze * MeSH
• osteokalcin metabolismus   MeSH
• proliferace buněk MeSH
• regenerace kostí * MeSH
• skot MeSH
• syrovátkové proteiny metabolismus   MeSH
• tkáňové inženýrství MeSH
• tuková tkáň cytologie   metabolismus   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• skot MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH

Nanostructuring of biomaterials is used to create an appropriate interfacial layer that promotes stable cellular adhesion, proliferation, and differentiation on orthopedic and dental implants. Here, we synthesize self-organized arrays of composite-oxide nano-mounds through anodizing Al/Ta bilayers sputtered on substrates to cover the "missing" smallest size range of 10-40 nm for structuring an advanced inorganic biomaterial-Al2 O3 -doped Ta2 O5 films. The osteoblast-like cells appear to be able to recognize the finest differences in the film nano-morphologies. In the absence of serum proteins, the adhesion and cell growth are substantially enhanced on the 20 and 40 nm nanoarrays while in complete medium the cells show better initial adhesion on the 10 nm nanoarrays. The proliferation assay reveals a significant rise in cell number on the 20 and 40 nm nanoarrays during the first 7 days. A remarkable increase in the alkaline phosphatase activity is noticed on the 40 nm nanoarray. Immunostaining of cells adhered to the nano-mound surfaces shows that the cells are well spread over all the nanostructured films with organized actin fibers. The larger surface areas and improved focal contacts are again associated with the 20 and 40 nm nanoarrays. The findings help improve compatibility of living cells with the metal-oxide nanostructured surfaces developed for tissue engineering. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 106B: 1645-1654, 2018.

• MeSH
• biokompatibilní materiály chemie   MeSH
• buněčná adheze MeSH
• buněčná diferenciace * MeSH
• buněčné linie MeSH
• lidé MeSH
• nanostruktury chemie   MeSH
• osteoblasty cytologie   metabolismus   MeSH
• oxid hlinitý chemie   MeSH
• poréznost MeSH
• proliferace buněk * MeSH
• tkáňové inženýrství metody   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH